Nucleic acid preservation solution and methods of manufacture and use

ABSTRACT

Disclosed is nucleic acid preserving compositions and methods of manufacturing and using the same. Compositions include a carrier, a chaotropic agent, a buffering agent, a chelating agent, a surfactant, an alcohol, an acid, and a mucolytic agent. Compositions as aqueous solutions can include water as a carrier. Preferred embodiments include water, guanidine thiocyanate, Tris, EDTA, SLS, SDA 3C, HCl, and N-acetyl-L-cysteine. Some embodiments include a colored dye as a visual indicator. Methods of manufacturing include combining the components into a mixture, such as an aqueous solution. Methods of use include providing a biological sample that includes nucleic acid and contacting the biological sample with the composition. Kits include the composition disposed in a portion of a biological sample collection apparatus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Prov. Pat.App. Ser. No. 62/446,818, filed Jan. 16, 2017, entitled “Nucleic AcidPreservation Solution and Methods of Manufacture and Use,” the entiretyof which is incorporated herein by reference. This application alsoclaims the benefit of and priority to: U.S. Prov. Pat. App. Ser. No.62/510,174, filed May 23, 2017; U.S. Prov. Pat. App. Ser. No.62/453,459, filed Feb. 1, 2017; U.S. Prov. Pat. App. Ser. No.62/513,235, filed May 31, 2017; U.S. Prov. Pat. App. Ser. No.62/512,594, filed May 30, 2017; U.S. Prov. Pat. App. Ser. No.62/529,355, filed Jul. 6, 2017; U.S. Prov. Pat. App. Ser. No.62/590,165, filed Nov. 22, 2017, the entirety of each of which isincorporated herein by reference.

BACKGROUND 1. Technical Field

The present disclosure relates to preserving nucleic acid. Specifically,the present disclosure relates to compositions and methods forpreserving human nucleic acid in a biological sample for furtheranalysis.

2. Related Technology

Nucleic acid can be extracted from biological samples that includecellular and/or cell-free nucleic acids. Extracted nucleic acid can beused for a variety of analytical purposes, including genealogicalanalysis. Extraction of nucleic acids from saliva can be particularlyuseful, as saliva sample collection is relatively non-invasive.

Nucleic acid-containing biological samples often need to be properlyprocessed for specific types of nucleic acid analysis. Analyticaltechniques such as nucleic acid sequencing (e.g., next generationsequencing (NGS)), for example, may require specific processing orpre-processing steps that depend on the specific platform to be used. Insome cases, the nucleic acid-containing biological samples may need tobe processed in order to stabilize the sample or nucleic acid thereof.Stabilizing solutions are often added to nucleic acid-containingbiological samples to ensure survival of a portion of the nucleic acidsuntil analysis thereof can be performed.

Existing stabilizing solutions may not be optimal for certain types ofanalytical techniques or devices for performing the same. For instance,a stabilizing solution formulated for optimal or suitable analysis in acertain next generation sequencer, may not be optimal or suitable foranalysis in other next generation sequencers. In some cases, improperformulation may produce or lead to analytical artifacts, high backgroundsignal (or noise), contamination and/or retention of microbial nucleicacids, and/or reduce the total potential yield of human nucleic acid.

Existing stabilizing solutions may also be deficient in controllingmicrobial life. Biological sample, such as saliva, often include and/orbecome contaminated with one or more microbes (e.g., bacteria, fungi,etc.). These microbes contain nucleic acids that may interfere or bedetected along with the nucleic acid of the host or source of thebiological sample. Preservation solutions may inadvertently stabilizemicrobial nucleic acids or even permit the growth of the microorganisms.

Accordingly, there continues to be a need for a universal nucleic acidstabilizing solution suitable for a variety of analytical techniques anddevices and/or a solution that provides a better overall yield ofnucleic acid and quality of sample, as compared to existing products.

BRIEF SUMMARY

Embodiments of the present disclosure solve one or more of the foregoingor other problems in the art with one or more embodiments comprising anucleic acid preservation, stabilization, and/or preparationcompositions, kits comprising the same, and methods of manufacturing andusing the same. For instance, some embodiments of the present disclosureinclude compositions for preserving, stabilizing, and/or preparingnucleic acid in a biological sample. The composition can be suitable foruse in a variety of analytical techniques and devices. The compositioncan yield high amounts of nucleic acid for subsequent analysis. Forexample, the composition can yield high amounts of human nucleic acid(e.g., DNA), preferably and/or optionally with low amounts of microbial(e.g., bacterial) nucleic acid (e.g., DNA) for subsequent analysis. Thecomposition can comprise a solution or water-based (e.g., aqueous)liquid, optionally (light) blue in color, suitable for use in thestabilization of human nucleic acid (DNA) and prevention of bacterialcontamination and for long term storage.

An embodiment of the present disclosure includes a nucleic acidpreservation composition, comprising an aqueous carrier, a chaotropicagent, a buffering agent, a chelating agent, a surfactant, an alcohol,an acid; and a mucolytic agent. An embodiment can further include avisual indicator. In some embodiments, the aqueous carrier can be orcomprise water, preferably filtered, purified, distilled, and/ordeionized water. In some embodiments, the chaotropic agent can be orcomprise guanidine and/or thiocyanate, preferably guanidine thiocyanate.In some embodiments, the buffering agent can be or comprisetris(hydroxymethyl)aminomethane (Tris), preferably Tris-HCl, morepreferably Trizma® base. In some embodiments, the chelating agent can beor comprise ethylenediaminetetraacetic acid (EDTA), preferably as EDTAdisodium salt, more preferably as EDTA disodium (salt) dihydrate. Insome embodiments, the surfactant can be or comprise sodium lauroylsarcosinate (SLS). In some embodiments, the alcohol can be or comprise aspecially denatured alcohol (SDA) or a mixture of ethanol andisopropanol, preferably a mixture of about 95% ethanol, v/v and about 5%isopropanol, v/v, or SDA 3C. In some embodiments, the acid can be orcomprise hydrochloric acid. In some embodiments, the mucolytic agent canbe or comprise N-acetyl-L-cysteine. In some embodiments, the visualindicator can be or comprise a coloring agent, such as a dye (e.g., FD&CBlue No. 1).

An embodiment of the present disclosure includes a human nucleic acidpreservation composition, comprising about 43.92% chaotropic agent(e.g., guanidine thiocyanate), w/w, about 2.65% buffering agent (e.g.,Tris), w/w, about 0.81% chelating agent (e.g., EDTA disodium dihydrate),w/w, about 0.279% surfactant (e.g., SLS), w/w, about 17.73% alcohol(e.g., SDA 3C), w/w, about 0.093% mucolytic agent (e.g.,N-acetyl-L-cysteine), w/w, about 0.4% acid (e.g., hydrochloric acid),w/w or acid qs to about pH 8.0; and/or about 34.12% carrier (e.g., anaqueous carrier comprising filtered, purified, distilled, and/ordeionized water) or carrier qs to 100%. An embodiment can furtherinclude about 0.00037%, w/w, visual indicator (e.g., FD&C Blue No. 1) orequivalent thereof (e.g., 0.185%, w/w, of a 0.2%, w/w, visual indicatorconcentrate (e.g., in water)).

One or more embodiments can include 43.92% chaotropic agent (e.g.,guanidine thiocyanate), w/w, ±10%, 2.65% buffering agent (e.g., Tris),w/w, ±10%, 0.81% chelating agent (e.g., EDTA or EDTA disodium (salt)dihydrate), w/w, ±10%, 0.279% surfactant (e.g., SLS), w/w, ±10%, 17.73%alcohol (e.g., SDA 3C or a mixture of 95% ethanol, v/v, ±10%, and 5%isopropanol, v/v, ±10%), w/w, ±10%, 0.093% mucolytic agent (e.g.,N-acetyl-L-cysteine), w/w, ±10%, and/or acid (e.g., hydrochloric acid)qs to pH 8.0, ±10%, with a carrier (e.g., an aqueous carrier, preferablyfiltered, purified, distilled, and/or deionized water) qs to 100%. Anembodiment can further include 0.00037%, w/w, ±10% visual indicator(e.g., FD&C Blue No. 1) or equivalent thereof (e.g., 0.185%, w/w, ±10%,of a 0.2%, w/w, ±10% visual indicator concentrate (e.g., in water)). Insome embodiments, the amount of each component, ±10%, is further(limited to the recited amount) ±9%, preferably ±8%, more preferably±7%, still more preferably ±6%, still more preferably ±5%, still morepreferably ±4%, still more preferably ±3%, still more preferably ±2%,still more preferably ±1%.

One or more embodiments can include 20-50% chaotropic agent, w/w, 0.1-5%buffering agent, w/w, 0.05-2.5% chelating agent, w/w, 0.01-5%surfactant, w/w, 5-25% alcohol, w/w, 0.005-0.25% mucolytic agent, w/w,0.005-5% acid or acid qs to pH 6.5-9.5, and/or 10-60% carrier or carrierqs to 100%. An embodiment can include 0.00005-0.5%, w/w, visualindicator (or 0.01-2.5%, w/w, of a 0.05-5%, w/w, visual indicatorconcentrate (e.g., in water)).

One or more embodiments can be (substantially) devoid of (additional orany) antimicrobial(s) (e.g., bactericidal and/or bacteriostatic)agent(s) (e.g., besides or other than the alcohol(s), chaotropicagent(s), surfactant(s)/detergent(s), and/or mucolytic agent(s)). One ormore embodiments can be (substantially) devoid of (additional or any)ribonuclease inhibitor(s), or inhibitor(s) of ribonuclease (e.g.,besides or other than the chaotropic agent(s)). One or more embodimentscan be (substantially) devoid of (any) a protease(s).

Some embodiments include a method of stabilizing nucleic acid. Themethod can include providing a biological sample containing the nucleicacid and combining a composition of the present disclosure with thebiological sample. The method can also include other processing stepsknown in the art. An embodiment of the present disclosure includes amethod of stabilizing nucleic acid (e.g., human nucleic acid, such ashuman DNA). An embodiment comprises contacting a biological samplecontaining the nucleic acid with a composition of the presentdisclosure. In an embodiment, the biological sample comprises humansaliva.

Some embodiments include a biological sample preservation kit. The kitcan comprise a sample collection apparatus and a nucleic acidpreservation composition. The sample collection apparatus can comprise asolution compartment. The nucleic acid preservation composition can bedisposed in the solution compartment. An embodiment of the presentdisclosure includes a kit comprising a composition of the presentdisclosure disposed in a portion of a sample collection apparatus.

Some embodiments include a method of manufacturing a composition of thepresent disclosure. The method can include combining components of thepresent disclosure. The method can also include other manufacturingsteps known in the art. An embodiment of the present disclosure includesa method of manufacturing a nucleic acid stabilization composition. Anembodiment comprises obtaining a carrier and adding to the carriercomponents or ingredients of a composition of the present disclosure.

Additional features and advantages of exemplary embodiments of thepresent disclosure will be set forth in the description which follows,and in part will be obvious from the description, or may be learned bythe practice of such exemplary embodiments. The features and advantagesof such embodiments may be realized and obtained by means of theinstruments and combinations particularly pointed out in the appendedclaims. These and other features will become more fully apparent fromthe following description and appended claims, or may be learned by thepractice of such exemplary embodiments as set forth hereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to describe the manner in which the above-recited and otheradvantages and features of the present disclosure can be obtained, amore particular description of the implementations briefly describedabove will be rendered by reference to specific implementations thereof,which are illustrated in the appended drawings. For betterunderstanding, the like elements have been designated by like referencenumbers throughout the figure(s). Understanding that these drawingsdepict only typical implementations of the invention and are nottherefore to be considered to be limiting of its scope, the inventionwill be described and explained with additional specificity and detailthrough the use of the accompanying drawing(s) in which:

FIG. 1A is an image of a gel with high molecular weight DNA preservedusing compositions according to an embodiment of the present disclosure;and

FIG. 1B is an image of a gel with Bionexus All Purpose HI-LO DNA Marker.

DETAILED DESCRIPTION

Before describing various embodiments of the present disclosure indetail, it is to be understood that this disclosure is not limited tothe specific parameters and description of the particularly exemplifiedsystems, methods, and/or products that may vary from one embodiment tothe next. Thus, while certain embodiments of the present disclosure willbe described in detail, with reference to specific features (e.g.,configurations, parameters, properties, steps, components, ingredients,members, elements, parts, and/or portions, etc.), the descriptions areillustrative and are not to be construed as limiting the scope of thepresent disclosure and/or the claimed invention. In addition, theterminology used herein is for the purpose of describing theembodiments, and is not necessarily intended to limit the scope of thepresent disclosure and/or the claimed invention.

While the detailed description is separated into sections, the sectionheaders and contents within each section are not intended to beself-contained descriptions and embodiments. Rather, the contents ofeach section within the detailed description are intended to be read andunderstood as a collective whole where elements of one section maypertain to and/or inform other sections. Accordingly, embodimentsspecifically disclosed within one section may also relate to and/orserve as additional and/or alternative embodiments in another sectionhaving the same and/or similar systems, devices, methods, and/orterminology.

Abbreviated List of Defined Terms

To assist in understanding the scope and content of the foregoing andforthcoming written description and appended claims, a select few termsare defined directly below. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which the presentdisclosure pertains.

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim, “and those that donot materially affect the basic and novel characteristic(s)” of theclaimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q.461, 463 (CCPA 1976) (emphasis in the original); see also MPEP §2111.03. Thus, the term “consisting essentially of” when used in a claimof this disclosure is not intended to be interpreted to be equivalent to“comprising.”

The term “nucleic acid” as used herein refers to a naturally occurringor synthetic oligonucleotide or polynucleotide, whether DNA or RNA orDNA-RNA hybrid, single-stranded or double-stranded, sense or antisense,which is capable of hybridization to a complementary nucleic acid byWatson-Crick base-pairing. Nucleic acids of the invention can alsoinclude nucleotide analogs (e.g., BrdU, dUTP, 7-deaza-dGTP), andnon-phosphodiester internucleoside linkages (e.g., peptide nucleic acid(PNA) or thiodiester linkages). In particular, nucleic acids caninclude, without limitation, DNA, RNA, cDNA, gDNA, ssDNA, dsDNA or anycombination thereof.

The term “sample,” “biological sample,” and the like refers to ananimal; a tissue or organ from an animal; a cell (either within asubject, taken directly from a subject, or a cell maintained in cultureor from a cultured cell line); a cell lysate (or lysate fraction) orcell extract; a solution containing one or more molecules derived from acell, cellular material, or viral material (e.g. a polypeptide ornucleic acid); or a solution containing a naturally or non-naturallyoccurring nucleic acid, which is or can be assayed as described herein.A sample may also be any bodily fluid or excretion that contains one ormore cells, cell components, or nucleic acids, including, but notlimited to cellular, nuclear, or cell-free nucleic acids.

By “bodily fluid” is meant a naturally occurring fluid, includingwithout limitation a liquid, semi-solid, aerated liquid, liquid-gasmixture, and so forth, from an animal (e.g., human or non-human animal).Such bodily fluids can include, but are not limited to, saliva, sputum,serum, plasma, blood, urine, mucus, perspiration, tears or otherophthalmic fluids, otic fluids, puss (e.g., from a blister or sore),gastric fluids or juices, fecal fluids, pancreatic fluids or juices,semen, products of lactation or menstruation, spinal fluid, fluid bonemarrow, or lymph.

By “sputum” is meant that mucoid matter contained in or discharged fromthe nasal or buccal cavity of a mammal. Sputum, as used herein,generally includes saliva and discharges from the respiratory passages,including the lungs.

By “saliva” is meant the secretion, or combination of secretions, fromany of the salivary glands, including the parotid, submaxillary, andsublingual glands, optionally mixed with the secretion from the buccalglands.

By “mucoid” is meant any bodily fluid containing mucin.

By “mucin” is meant any mucoprotein that raises the viscosity of themedium surrounding the cells that secrete it.

As used herein, the term “about,” with regard to a value, means+/−10% ofthe stated value or amount represented thereby. For instance, throughoutthe present disclosure, the term “about” is used in connection with apercent concentration or composition of a component or ingredient (e.g.,in a mixture, such as a fluid or liquid mixture, aqueous mixture,solution, etc., optionally or preferably measured as a w/w percent, w/vpercent, v/v percent, etc.). In such instance, the term “about” and/orthe term “+/−10%” implies and/or includes +/−10% of the stated numericvalue, as opposed to +/−10 percentage points of the recited percent. Byway of example, where 20% w/w of a component or ingredient reflects 20 gof the component or ingredient per 100 mL of total mixture, the term“about” and/or the term “+/−10%” implies and/or includes a recited rangefrom 18 g to 22 g (i.e., from 18% w/w to 22% w/w), not a range of 10%w/w to 30% w/w. Alternatives for so-called “about” values and/or +/−10%include +/−1%, +/−2%, +/−3%, +/−4%, +/−5%, +/−6%, +/−7%, +/−8%, or +/−9%of the stated value, each of which is contemplated as a suitablealternative to or substitute for the term “about” or the use of +/−10%herein.

As used herein, the terms “approximately” and “substantially” representor imply an (or any) amount close to the stated amount (e.g., that stillperforms a desired function or achieves a (desired or expected) result).For example, the terms “approximately” and “substantially” may refer toan amount that is within, or less than, 10%, 5%, 1%, 0.1%, 0.01%, orother percent of a stated amount. As used herein, the term“substantially devoid” means (1) an undetectable or unquantifiableamount, (2) less than or below an amount generally considered by thoseskilled in the art to reflect a detectable or quantifiable amount,and/or (3) less than or below an amount generally considered by thoseskilled in the art to be functional or able to achieve a (desired orexpected) result (e.g., less than 10%, 5%, 1%, 0.1%, 0.01%, or otherpercent).

By “Quantum satis” (also referred to as “q.s.” or “qs”) is meant theamount that is enough. Accordingly, a component or ingredient “qs 100%,”“provided at qs 100%,” or “qs to 100%” indicates that the component oringredient is provided or included in an amount sufficient to completethe composition or to bring the total (of all components, whetherrecited or not) to 100%. It is noted, however, that a (final) componentor ingredient “qs 100%,” “provided at qs 100%,” or “qs to 100%” does notindicate that the mixture consists of, consists essentially of, or onlycontains the components listed or recited immediately before the “qs100%” component. In other words, “qs 100%,” and similar terms, is meantto be an open-ended expression indicating the source of the remainder,whatever that remainder may be.

By “alcohol” is meant a water-miscible organic compound containing ahydroxyl group, including water-miscible mixtures of hydroxyl-containingorganic compounds.

By “aqueous” is meant a medium or matter that contains 30% or more water(by volume or by weight).

By “aqueous solution” is meant a solution or suspension that contains30% or more water by volume.

By “denaturing agent” is meant a substance that alters the natural stateof that to which it is added.

By “chaotropic agent” is meant a molecule that exerts chaotropicactivity. As understood by those skilled in the art, molecules thatexert chaotropic activity may disrupt the hydrogen-bonding networkbetween water molecules, thereby affecting the stability of the nativestate of other molecules (in the solution), mainly macromolecules(proteins, nucleic acids) by weakening the hydrophobic effect.Accordingly, molecules that exert chaotropic activity may haveprotein-denaturing activity (or be protein denaturants).

By “antimicrobial agent” is meant a substance or group of substanceswhich reduces the rate of growth of an organism compared to the rate ofgrowth of the organism in their absence. A reduction in the rate ofgrowth of an organism may be by at least 5%, more desirably, by at least10%, even more desirably, by at least 20%, 50%, or 75%, and mostdesirably, by 90% or more. The definition also extends to substanceswhich affect the viability, virulence, or pathogenicity of an organism.An antimicrobial agent can be natural (e.g., derived from bacteria orother source), synthetic, or recombinant. An antimicrobial agent can bebacteriostatic, bactericidal or both. An antimicrobial agent isbacteriostatic if it inhibits cell division without affecting theviability of the inhibited cell. An antimicrobial agent is bactericidalif it causes cell death. Cell death is commonly detected by the absenceof cell growth in liquid growth medium (e.g., absence of turbidity) oron a solid surface (e.g., absence of colony formation on agar). Those ofskill in the art know that a substance or group of substances which isbacteriostatic at a given concentration may be bactericidal at a higherconcentration. Certain bacteriostatic substances are not bactericidal atany concentration.

As used herein, “acetylcysteine” or “N-acetylcysteine” (NAC), includesany form of acetylcysteine, including N-acetyl-L-cysteine,N-acetyl-D-cysteine, and racemic N-acetylcysteine or a (racemic) mixtureof N-acetyl-L-cysteine and N-acetyl-D-cysteine). Reference to one formof acetylcysteine supports a specific reference to any form ofacetylcysteine.

As used herein, the term “composition” includes products, formulations,and mixtures, as well as devices, apparatus, assemblies, kits, and soforth. Similarly, the term “method” includes processes, procedures,steps, and so forth.

Various aspects of the present disclosure, including systems, methods,and/or products may be illustrated with reference to one or moreembodiments or implementations, which are exemplary in nature. As usedherein, the terms “embodiment” and “implementation” mean “serving as anexample, instance, or illustration,” and should not necessarily beconstrued as preferred or advantageous over other aspects disclosedherein. In addition, reference to an “implementation” of the presentdisclosure or invention includes a specific reference to one or moreembodiments thereof, and vice versa, and is intended to provideillustrative examples without limiting the scope of the invention, whichis indicated by the appended claims rather than by the descriptionthereof.

As used herein, a “feature” of the present disclosure or embodimentdisclosed herein refers to a property, component, ingredient, element,part, portion, (method) step, or other aspect of the subject matter athand.

As used throughout this disclosure, the words “can” and “may” are usedin a permissive sense (i.e., meaning having the potential to), ratherthan the mandatory sense (i.e., meaning must). Additionally, the terms“including,” “having,” “involving,” “containing,” “characterized by,”variants thereof (e.g., “includes,” “has,” and “involves,” “contains,”etc.), and similar terms as used herein, including the claims, shall beinclusive and/or open-ended, shall have the same meaning as the word“comprising” and variants thereof (e.g., “comprise” and “comprises”),and do not exclude additional, un-recited elements or method steps,illustratively.

The word “or” as used herein means any one member of a particular listand also includes any combination of members of that list.

As used in this specification and the appended claims, the singularforms “a,” “an” and “the” each contemplate, include, and specificallydisclose both the singular and plural referents, unless the contextclearly dictates otherwise. For example, reference to a “protein”contemplates and specifically discloses one, as well as two or moreproteins. Similarly, use of a plural referent does not necessarilyrequire a plurality of such referents, but contemplates, includes, andspecifically discloses one, as well as two or more of such referents,unless the context clearly dictates otherwise.

It is noted that embodiments of the present disclosure can comprise oneor more combinations of two or more of the features described herein. Asused herein, “feature(s)” and similar terms can include, for example,compositions, ingredients, components, elements, members, parts,portions, systems, methods, configurations, parameters, properties, andso forth. Embodiments can include any of the features, options, and/orpossibilities set out elsewhere in the present disclosure, including inother aspects or embodiments of the present disclosure. It is also notedthat each of the foregoing, following, and/or other features describedherein represents a distinct embodiment of the present disclosure.Features can also be combined and/or combinable with another one or moreother features in any suitable combination and/or order, with or withoutone or more additional features included therewith or performedtherebetween, to form unique embodiments, each of which is contemplatedin the present disclosure. Such combinations of any two or more of suchfeatures represent distinct embodiments of the present disclosure.Accordingly, the present disclosure is not limited to the specificcombinations of exemplary embodiments described in detail herein anddisclosure of certain features relative to a specific embodiment of thepresent disclosure should not be construed as limiting application orinclusion of said features to the specific embodiment.

In addition, unless a feature is described as being requiring in aparticular embodiment, features described in the various embodiments canbe optional and may not be included in other embodiments of the presentdisclosure. Moreover, unless a feature is described as requiring anotherfeature in combination therewith, any feature herein may be combinedwith any other feature of a same or different embodiment disclosedherein. Likewise, any steps recited in any method described hereinand/or recited in the claims can be executed in any suitable order andare not necessarily limited to the order described and/or recited,unless otherwise stated (explicitly or implicitly). Such steps can,however, also be required to be performed in a particular order incertain embodiments of the present disclosure.

It will also be appreciated that where two or more values, or a range ofvalues (e.g., less than, greater than, at least, and/or up to a certainvalue, and/or between two recited values) is disclosed or recited, anyspecific value or range of values falling within the disclosed values orrange of values is likewise specifically disclosed and contemplatedherein. Thus, disclosure of an illustrative measurement (e.g., length,width, thickness, etc.) that is less than or equal to about 10 units orbetween 0 and 10 units includes, illustratively, a specific disclosureof: (i) a measurement of 9 units, 5 units, 1 units, or any other valuebetween 0 and 10 units, including 0 units and/or 10 units; and/or (ii) ameasurement between 9 units and 1 units, between 8 units and 2 units,between 6 units and 4 units, and/or any other range of values between 0and 10 units.

To facilitate understanding, like references (i.e., like naming ofcomponents and/or elements) have been used, where possible, to designatelike elements common to different embodiments of the present disclosure.Similarly, like components, or components with like functions, will beprovided with similar reference designations, where possible. Specificlanguage will be used herein to describe the exemplary embodiments.Nevertheless it will be understood that no limitation of the scope ofthe disclosure is thereby intended. Rather, it is to be understood thatthe language used to describe the exemplary embodiments is illustrativeonly and is not to be construed as limiting the scope of the disclosure(unless such language is expressly described herein as essential).

Illustrative Embodiments

The following description of embodiments includes disclosure that isrelevant to one or more embodiments of the present disclosure.Accordingly, some embodiments can include features disclosed in thefollowing examples without departing from the scope of the presentdisclosure. In other words, features disclosed in the following examplescan be included and/or incorporated into any one or more of theembodiments disclosed herein.

Compositions

Some embodiments of the present disclosure include a composition. Thecompositions can render sputum or saliva as a viable source of nucleicacids for purification and analysis. The compositions provide theadvantageous properties of chemical stabilization of nucleic acids andthe inhibition of nucleases, including deoxyribonucleases, and microbialgrowth. Chemical stabilization of the nucleic acids in a saliva samplecan be achieved through the use of buffers, acids, chelating agents,mucolytic agents, chaotropic agents, surfactants, and alcohol.

The compositions of the present disclosure, when mixed with a biologicalsample, e.g., mucin-containing bodily fluid, can preserve the nucleicacids at room temperature under ambient conditions for extended periodsof time. Samples can also be refrigerated, but freezing of the samplesbefore nucleic acid recovery and purification is not required. Theproperties of certain composition of the present disclosure are that it(a) chemically stabilizes nucleic acids, (b) inhibits nucleases that maybe present in the saliva, and (c) is compatible with proteolytic enzymesand other reagents used to purify/amplify oligo- or polynucleotides.

Carriers

In at least one embodiment, the composition can include a carrier.Preferably, the carrier can be a liquid carrier or solvent, morepreferably an aqueous carrier or solvent, still more preferably water.Most preferably, the carrier can be or comprise purified, filtered(e.g., 0.2 micron filtered), distilled, and/or deionized water.Accordingly, the composition can include a carrier. The carrier can beor comprise water, such as filtered water, purified water, distilledwater, or deionized water.

In some embodiments, the composition can include a carrier qs to 100%.In some embodiments, the composition can include 10-60%, preferably15-55%, more preferably 20-50%, still more preferably 25-45% still morepreferably 28-40%, still more preferably 30-35%, still more preferably31-34%, still more preferably 32-33% carrier, w/w (or any value or rangeof values therebetween). Most preferably, the composition can include(about) 32.602% water, w/w.

Chaotropic Agents

The composition can include one or more chaotropic agents. In one ormore embodiments, the chaotropic agent(s) can be a protein denaturant.In some embodiments, the chaotropic agent can be selected from the groupconsisting of: guanidinium chloride and/or guanidinium thiocyanate.Accordingly, in at least one embodiment, the composition can include achaotropic agent. Preferably, the chaotropic agent can be or compriseguanidine (or guanidinium) or a suitable salt thereof. More preferably,the chaotropic agent can be or comprise guanidine thiocyanate. In atleast one embodiment, the chaotropic agent can be or comprisethiocyanate. In at least one embodiment, the chaotropic agent can be orcomprise guanidine isothiocyanate, guanidine chloride, guanidinehydrochloride, guanidinium iodide, and so forth.

In some embodiments, the chaotropic agent can be in, have, comprise, orbe provided in a dry, solid, powdered, anhydrous, and/or granular form.In some embodiments, the chaotropic agent can have a purity of at least,up to, and/or about 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%. 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (as measured by a suitablematerial assay, such as CoA). In some embodiments, the chaotropic agentcan comprise or be (provided) in the form of a stock solution (e.g., inwater) having any suitable concentration. In some embodiments, thechaotropic agent can have a purity substantially corresponding to theconcentration of the chaotropic agent in solution (as measured by asuitable material assay, such as CoA).

In some embodiments, the composition can include 20-50%, preferably25-49%, more preferably 30-48% still more preferably 35-47%, still morepreferably 40-46%, still more preferably 42-45%, still more preferably43-44% of the chaotropic agent (e.g., guanidine thiocyanate), w/w, orany value or range of values therebetween. Most preferably, thecomposition can include (about) 43.92% guanidine thiocyanate, w/w. Thechaotropic agent (e.g., guanidine thiocyanate) can be included in thecomposition at about 43.92% w/w, or in a range of about 35% to about50%, preferably about 40% to about 46%, more preferably about 42% toabout 45%, still more preferably about 43% to about 44%, w/w.

Buffering Agents

The composition can include one or more buffering agents (or buffers, pHbuffers, etc.). Examples of buffering agents include, but are notlimited to tris(hydroxymethyl)aminomethane (also known as Tris; Trisbase, 2-Amino-2-(hydroxymethyl)-1,3-propanediol, THAM, Trometamol) or asuitable formulation thereof (e.g., tris(hydroxymethyl)aminomethanehydrochloride, or Tris-HCl,), Trizma® base (e.g., Tris 40% (w/w) stocksolution in water), HEPES, BES, MOPS, HEPES, TAE, TBE, phosphate buffer,sodium borate buffer, sodium cacodylate buffer, and so forth.Preferably, the buffering agent can be or comprisetris(hydroxymethyl)aminomethane (Tris). More preferably, the bufferingagent can be or comprise Tris-HCl. Most preferably, the buffering agentcan be or comprise Trizma® base.

In some embodiments, the buffering agent can be in, have, comprise, orbe provided in a dry, solid, powdered, anhydrous, and/or granular form.In some embodiments, the buffering agent can have a purity of at least,up to, and/or about 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%. 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (as measured by a suitablematerial assay, such as CoA). In some embodiments, the buffering agentcan comprise or be (provided) in the form of a stock solution (e.g., inwater) having any suitable concentration (e.g., Tris ˜40% (w/w) stocksolution in water). In some embodiments, the buffering agent can have apurity substantially corresponding to the concentration of the bufferingagent in solution (as measured by a suitable material assay, such asCoA).

The buffering agent can be included in the composition at about 2.65% %w/w, or in a range of about 0.1% to about 5%, preferably about 0.5% toabout 4.5%, more preferably about 0.75% to about 4%, still morepreferably about 1% to about 3.5%, still more preferably about 1.5% toabout 3.25%, still more preferably about 2% to about 3%, still morepreferably about 2.5% to about 2.8%, w/w. In some embodiments, thecomposition can include 1-5%, preferably 1.25-4.5%, more preferably1.5-4% still more preferably 1.75-3.75%, still more preferably 2-3.5%,still more preferably 2.25-3%, still more preferably 2.5-2.75% of thebuffering agent (e.g., Tris), w/w, or any value or range of valuestherebetween. Most preferably, the composition can include (about) 2.65%Tris, w/w.

Chelating Agents

In at least one embodiment, the composition can include a chelatingagent (or chelator). Preferably, the chelating agent can be or compriseethylenediaminetetraacetic acid (EDTA) or suitable salt and/or hydratethereof. More preferably, the chelating agent can be or comprise, or beprovided as EDTA disodium salt. Still more preferably, the chelatingagent can be or comprise, or be provided as EDTA disodium (salt)dihydrate. In at least one embodiment, the chelating agent can be orcomprise ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraaceticacid (EGTA), nitrilotriacetic acid (NTA), an ethylenediamine (or1,2-diaminoethane), and so forth. In some embodiments, the chelatingagent comprises, includes, or is provide with a counter ion (e.g.,sodium). In at least one embodiment, the chelating agent comprises,includes, or is provide as a hydrate (e.g., dihydrate).

The composition can include one or more chelating agents. The chelatingagent of the composition can be selected from the group consisting of:ethylenediamine tetraacetic acid (EDTA), cyclohexane diaminetetraacetate(CDTA), diethylenetriamine pentaacetic acid (DTPA),tetraazacyclododecanetetraacetic acid (DOTA),tetraazacyclotetradecanetetraacetic acid (TETA), desferrioximine,nitrilotriacetic acid (NTA), an ethylenediamine (or 1,2-diaminoethane),or respective chelator analogs, salts, and/or hydrates thereof.Preferably, the chelating agent can be or comprise EDTA (e.g., as EDTAdisodium salt, preferably as EDTA disodium (salt) dihydrate). In someembodiments, the chelating agent comprises, includes, or is provide witha counter ion (e.g., sodium). In at least one embodiment, the chelatingagent comprises, includes, or is provide as a hydrate (e.g., dihydrate).

In some embodiments, the chelating agent can be in, have, comprise, orbe provided in a dry, solid, powdered, anhydrous, and/or granular form.In some embodiments, the chelating agent can have a purity of at least,up to, and/or about 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%. 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (as measured by a suitablematerial assay, such as CoA). In some embodiments, the chelating agentcan comprise or be (provided) in the form of a stock solution (e.g., inwater) having any suitable concentration. In some embodiments, thechelating agent can have a purity substantially corresponding to theconcentration of the chelating agent in solution (as measured by asuitable material assay, such as CoA).

The chelating agent (e.g., EDTA) can be included in the composition atabout 0.81% w/w, or in a range of about 0.05% to about 2.5%, preferablyabout 0.1% to about 2%, more preferably about 0.5% to about 1%, stillmore preferably about 0.75% to about 0.9%, w/w. In some embodiments, thecomposition can include 0.05-2.5%, preferably 0.1-2.25%, more preferably0.25-2% still more preferably 0.5-1.75%, still more preferably 0.6-1.5%,still more preferably 0.7-1.25%, still more preferably 0.75-1% of thechelating agent (e.g., EDTA), w/w, or any value or range of valuestherebetween). Most preferably, the composition can include (about)0.81% EDTA or EDTA disodium (salt) dihydrate, w/w.

Surfactants

In at least one embodiment, the composition can include a surfactant ordetergent. Preferably, the surfactant can be or comprise a lauroylsarcosinate. More preferably, the surfactant can be or comprise sodiumlauroyl sarcosinate (SLS). In at least one embodiment, the surfactantcan be or comprise one or more components selected from the groupconsisting of sodium dodecyl sulfate (SDS), polysorbates (Tween™),lauryl dimethyl amine oxide, cetyltrimethylammonium bromide (CTAB),polyethoxylated alcohols, polyoxyethylene sorbitan, octoxynol (TritonX100™), N,N-dimethyldodecylamine-N-oxide, hexadecyltrimethylammoniumbromide (HTAB), polyoxyl 10 lauryl ether, Bile salts (sodiumdeoxycholate, sodium cholate), polyoxyl castor oil (Cremophor™),nonylphenol ethoxylate (Tergitol™), cyclodextrins, lecithin,methylbenzethonium chloride (Hyamine™), and so forth. The compositioncan include a surfactant or detergent, such as urea, perchlorate,(sodium) dodecyl sulfate (SDS), and/or (sodium) lauroyl sarcosinate(SLS), preferably sodium lauroyl sarcosinate (SLS). In some embodiments,SLS can be preferable over SDS or other (less soluble) surfactants.

In some embodiments, the surfactant can be in, have, comprise, or beprovided in a dry, solid, powdered, anhydrous, and/or granular form. Insome embodiments, the surfactant can have a purity of at least, up to,and/or about 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%. 99.3%, 99.4%,99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (as measured by a suitable materialassay, such as CoA). In some embodiments, the surfactant can comprise orbe (provided) in the form of a stock solution (e.g., in water) havingany suitable concentration (e.g., about 10%, 15%, 20%, 25%, 28%, 29%,30%, 32%, 35%, 40%, or 45%, w/w, aqueous solution (e.g., in water). Insome embodiments, the surfactant can have a purity substantiallycorresponding to the concentration of the surfactant in solution (e.g.,about 30%, w/w) (as measured by a suitable material assay, such as CoA).

In some embodiments, the surfactant (e.g., SLS) can be included in thecomposition at about 0.279%, w/w. In some embodiments, the surfactantcan be included in the composition in a range of about 0.01% to about5%, w/w, preferably about 0.025% to about 2.5%, w/w, more preferablyabout 0.05% to about 2%, w/w, still more preferably about 0.075% toabout 1.5%, w/w, still more preferably about 0.1% to about 1%, w/w,still more preferably about 0.15% to about 0.5%, w/w, still morepreferably about 0.2% to about 0.4%, w/w, still more preferably about0.25% to about 0.3%, w/w. Some embodiments include 0.01% to 5%, w/w,preferably 0.025% to 2.5%, w/w, more preferably 0.05% to 2%, w/w, stillmore preferably 0.075% to 1.5%, w/w, still more preferably 0.1% to 1%,w/w, still more preferably 0.15% to 0.5%, w/w, still more preferably0.2% to 0.4%, w/w, still more preferably 0.25% to 0.3%, w/w, mostpreferably 0.279%, w/w, surfactant or SLS. In at least one embodiment,the surfactant (e.g., SLS) can be included in the composition at about0.93% w/w, of a ˜30% stock (aqueous) solution, or equivalent thereof.

Alcohols

In at least one embodiment, the composition can include an alcohol.Preferably, the alcohol can be or comprise ethanol. More preferably, thealcohol can be or comprise a mixture of ethanol and one or moreadditional chemicals or components. In at least one embodiment, the oneor more additional chemicals or components can be or compriseisopropanol. Still more preferably, the alcohol can be or comprise amixture of ethanol and isopropanol. In at least one embodiment, the oneor more additional chemicals or components can be or comprise methanol,propanol, butanol, isobutanol, and so forth. In at least one embodiment,the alcohol can be or comprise a specially denatured alcohol (SDA). Morepreferably, the alcohol can be or comprise SDA 3C, as known to thoseskilled in the art to comprise a mixture of about 95% ethanol v/v andabout 5% isopropanol v/v. The composition can include an alcohol, suchas ethanol, methanol, propanol, and/or isopropanol, preferably aspecially denatured alcohol (SDA) or a mixture of ethanol and anotheralcohol, such as methanol, n-propanol, isopropanol, n-butanol,trifluoroethanol, phenol, or 2,6-di-tert-butyl-4-methylphenol, morepreferably a mixture of ethanol and isopropanol, still more preferably amixture of ethanol and one or more additional chemicals or components,such as isopropanol.

In some embodiments, the surfactant can be in, have, comprise, or beprovided in a dry, solid, powdered, anhydrous, and/or granular form. Insome embodiments, the alcohol can be in, have, comprise, or be providedin a liquid, aqueous, and/or solution form. In some embodiments, thealcohol can comprise or be (provided) in the form of a stock solution(e.g., in water) having any suitable concentration of alcohol (e.g., inwater). In some embodiments, the alcohol can be substantially pure, or amixture of substantially pure alcohols. In some embodiments, the alcoholcan have a purity of at least, up to, and/or about 90%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%. 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or99.9% (or pure ethyl alcohol, 200 proof) (as measured by a suitablematerial assay, such as CoA).

In some embodiments, the alcohol can be or comprise a mixture or stocksolution of or comprising about 95% v/v ethanol and about 5% v/visopropanol. In some embodiments, the alcohol can be or comprise amixture or stock solution of or comprising 90-99% v/v ethanol and about1-10% v/v isopropanol. In certain embodiments, the alcohol can comprisea mixture of 50-99% ethanol v/v and 1-50% isopropanol v/v. Morepreferably, the alcohol can comprise a mixture of 60-98% ethanol v/v and2-40% isopropanol v/v. Still more preferably, the alcohol can comprise amixture of 75-97% ethanol v/v and 3-25% isopropanol v/v. Still morepreferably, the alcohol can comprise a mixture of 80-96% ethanol v/v and4-20% isopropanol v/v. Still more preferably, the alcohol can comprise amixture of 85-95% ethanol v/v and 5-15% isopropanol v/v. Still morepreferably, the alcohol can comprise a mixture of 90-95% ethanol v/v and5-10% isopropanol v/v. Still more preferably, the alcohol can comprise amixture of 92-95% ethanol v/v and 5-8% isopropanol v/v. Still morepreferably, the alcohol can comprise a mixture of 95% ethanol v/v and 5%isopropanol v/v. Most preferably, the alcohol can be or comprise SDA 3C.

The alcohol (e.g., SDA 3C) can be included in the composition at about17.73% w/w, or in a range of about 10% to about 25%, preferably about12% to about 22%, more preferably about 15% to about 20%, still morepreferably about 16% to about 19%, still more preferably about 17% toabout 18%, w/w. In some embodiments, the amount of alcohol included inthe composition can be less (e.g., about 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, or 60% less) than typical, traditional, or existing nucleicacid preservation solutions (e.g., making the composition more amendableto shipping or transport). In some embodiments, the composition caninclude 5-25%, preferably 10-22%, more preferably 12-20% still morepreferably 15-19%, still more preferably 16-18.5%, still more preferably17-18.25%, still more preferably 17.5-18% alcohol, w/w, or any value orrange of values therebetween.

Preferably, the alcohol comprises a mixture of ethanol and one or moreadditional chemicals or components, such as isopropanol, more preferablya mixture of about 95% ethanol, v/v and about 5% isopropanol, v/v. Stillmore preferably, the alcohol is a specially denatured alcohol (SDA),still more preferably SDA 3C (i.e., a mixture of ˜95% ethanol and ˜5%isopropanol, v/v). Most preferably, the composition can include (about)17.73% SDA 3C, w/w. In some embodiments, the alcohol (e.g., SDA 3C) canbe included in the composition at about 16.84% w/w, ethanol or in arange of about 10% to about 25%, preferably about 12% to about 22%, morepreferably about 15% to about 20%, still more preferably about 16% toabout 18%, still more preferably about 16.5% to about 17%, w/w, ethanol,and about 0.89% w/w, isopropanol or in a range of about 0.05% to about2.5%, preferably about 0.1% to about 2%, more preferably about 0.5% toabout 1.5%, still more preferably about 0.75% to about 1.25%, still morepreferably about 0.8% to about 1%, w/w, isopropanol.

In some embodiments, the amount of alcohol included in the compositioncan be less (e.g., about 50% less) than typical, traditional, orexisting nucleic acid preservation solutions (e.g., making thecomposition more amendable to shipping or transport).

Acids

In at least one embodiment, the composition can include an acid.Preferably, the acid can be or comprise hydrochloric acid (HCl). In atleast one embodiment, the acid can be or comprise hydrobromic acid(HBr), perchloric acid (HClO4), nitric acid (HNO3), or sulfuric acid(H2SO4). In at least one embodiment, the acid can be or comprisecarbonic acid (H2CO3) or acetic acid (CH3COOH). In at least oneembodiment, the acid can be or comprise phosphoric acid (H3PO4), boricacid (H3BO3), or Emerald Safe acid (ESA), and so forth.

In some embodiments, the acid can be in, have, comprise, or be providedin a dry, solid, powdered, anhydrous, and/or granular form. In someembodiments, the acid can have a purity of at least, up to, and/or about90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%. 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, or 99.9% (as measured by a suitable material assay, suchas CoA). In some embodiments, the acid can comprise or be (provided) inthe form of a stock solution (e.g., in water) having any suitableconcentration (e.g., about 10%, 15%, 20%, 25%, 30%, 32%, 35%, 37%, 38%,40%, or 45%, w/w, aqueous solution (e.g., in water). In someembodiments, the acid can have a purity substantially corresponding tothe concentration of the acid in solution (e.g., about 37%, w/w) (asmeasured by a suitable material assay, such as CoA).

In some embodiments, the composition can include acid (e.g.,hydrochloric acid), qs to pH about 8.0, or pH 7.5-9.5, pH 6.5-9.5, pH7-9, pH 7.2-8.8, pH 7.4-8.6, pH 7.5-8.5, pH 7.6-8.4, or pH 7.8-8.2 (orany value or range of values therebetween). In some embodiments, the pHof the composition can be greater than about 5 and less than about 12,preferably within a pH range of about 6 to about 11, more preferablywithin a pH range of about 6 to about 10, still more preferably within apH range of about 7 to about 9, still more preferably within a pH rangeof about 7.5 to about 8.5, and most preferably, with a pH of about 8.0.

In some embodiments, the acid (e.g., HCl) can be included in thecomposition at about 0.4% w/w, or in a range of about 0.01% to about 5%,preferably about 0.025% to about 2.5%, more preferably about 0.05% toabout 2%, more preferably about 0.1% to about 1.5%, more preferablyabout 0.25% to about 1%, more preferably about 0.5% to about 0.75%, morepreferably about 0.3% to about 0.5%, w/w. In some embodiments, thecomposition can include 0.005-5%, preferably 0.01-2.5%, more preferably0.025-1.5%, still more preferably 0.05-1% still more preferably0.1-0.75%, still more preferably 0.25-0.5% acid (e.g., hydrochloricacid), w/w. In at least one embodiment, the acid (e.g., HCl) can beincluded in the composition at about 1.08%, w/w, of a ˜37%, w/w, or ˜12Mstock (aqueous) solution, or equivalent thereof. Most preferably, thecomposition can include (about) 1.08% hydrochloric acid 37%, w/w, orequivalent thereof, or hydrochloric acid qs to pH (about) 8.0.

Without being bound to any theory, it is noted, and those skilled in theart will appreciate that different acids have different “strengths” orthe ability or tendency of the acid to lose a proton (H+). A strong acidis one that completely ionizes (dissociates) in a solution (providedthere is sufficient solvent). In water, for example, one mole of astrong acid HA dissolves yielding one mole of H+(as hydronium ion H3O+and higher aggregates) and one mole of the conjugate base, A−.Essentially, none of the non-ionized acid HA remains. Some examples ofstrong acids are hydrochloric acid (HCl), hydroiodic acid (HI),hydrobromic acid (HBr), perchloric acid (HClO4), nitric acid (HNO3) andsulfuric acid (H2SO4). In aqueous solution, each of these essentiallyionizes 100%. In contrast, a weak acid only partially dissociates.Examples in water include carbonic acid (H2CO3) and acetic acid(CH3COOH). At equilibrium, both the acid and the conjugate base arepresent in solution. Stronger acids have a larger acid dissociationconstant (Ka) and a smaller logarithmic constant (pKa=−log Ka) thanweaker acids. The stronger an acid is, the more easily it loses aproton, H+. Two key factors that contribute to the ease of deprotonationare the polarity of the H-A bond and the size of atom A, whichdetermines the strength of the H-A bond. Acid strengths also depend onthe stability of the conjugate base.

In light of the foregoing, the w/w amount of each acid necessary tobring the pH of the composition to a desired level is different. Forinstance, while (about) 1.08% hydrochloric acid 37%, w/w (in water), maybe sufficient to bring certain embodiments of the present disclosure topH (about) 8.0, 1.08% acetic acid 37%, w/w (in water), may be too weakto bring a similar embodiment to pH (about) 8.0, 1.08% sulfuric acid37%, w/w (in water), may be too strong to bring the embodiment to pH(about) 8.0, 1.08% nitric acid 37%, w/w (in water), may oxidize thealcohol, and so forth. Without being bound to any theory, even those ofordinary skill in the art may not, with further experimentation, be ableto determine which acids are suitable in one or more embodiments of thepresent disclosure.

Mucolytic Agents

In at least one embodiment, the composition can include a mucolyticagent. In one or more embodiments, the mucolytic agent can be orcomprise a reducing agent. Preferably, the mucolytic agent can be orcomprise an acetylcysteine (i.e., N-acetylcysteine (NAC), includingN-acetyl-L-cysteine, N-acetyl-D-cysteine, and racemic N-acetylcysteineor a (racemic) mixture of N-acetyl-L-cysteine and N-acetyl-D-cysteine).More preferably, the mucolytic agent can be or compriseN-Acetyl-L-cysteine. In at least one embodiment, the mucolytic agent canbe or comprise N-acetylcysteine (N-acetyl-L-cysteine), ascorbic acid,dithionite, erythiorbate, cysteine, glutathione, dithiothreitol,2-mercaptoethanol, dierythritol, a resin-supported thiol, aresin-supported phosphine, vitamin E, and/or trolox, or salts thereof,sodium citrate, potassium citrate, potassium iodide, ammonium chloride,guaiphenesin (or guaifenesin), Tolu balsam, Vasaka, ambroxol,carbocisteine, erdosteine, mecysteine, dornase alfa, and so forth. Thecomposition can include one or more mucolytic agent. Preferably, themucolytic agent is ascorbic acid, erythiorbate, N-acetylcysteine,dithiothreitol, or 2-mercaptoethanol, and most preferably, the mucolyticagent is N-acetylcysteine.

In one or more embodiments, the composition does not contain ascorbicacid, dithionite, erythiorbate, dithiothreitol, 2-mercaptoethanol,dierythritol, a resin-supported thiol, a resin-supported phosphine,vitamin E, trolox, and/or salts thereof. At least one embodiment is(substantially) devoid of ascorbic acid, dithionite, erythiorbate,dithiothreitol, 2-mercaptoethanol, dierythritol, a resin-supportedthiol, a resin-supported phosphine, vitamin E, trolox, and/or saltsthereof. At least one embodiment is (substantially) devoid of amucolytic agent besides N-acetyl-L-cysteine.

In some embodiments, the mucolytic agent can be in, have, comprise, orbe provided in a dry, solid, powdered, anhydrous, and/or granular form.In some embodiments, the mucolytic agent can have a purity of at least,up to, and/or about 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%. 99.3%,99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (as measured by a suitablematerial assay, such as CoA). In some embodiments, the mucolytic agentcan comprise or be (provided) in the form of a stock solution (e.g., inwater) having any suitable concentration. In some embodiments, themucolytic agent can have a purity substantially corresponding to theconcentration of the mucolytic agent in solution (as measured by asuitable material assay, such as CoA).

The mucolytic agent (e.g., N-acetylcysteine) can be included in thecomposition at about 0.093% w/w, or in a range of about 0.01% to about0.5%, preferably about 0.025% to about 0.25%, more preferably about0.05% to about 0.2%, still more preferably about 0.075% to about 0.15%,still more preferably about 0.08% to about 0.1%, w/w.

In some embodiments, the composition can include 0.005-0.25%, preferably0.005-0.2%, more preferably 0.01-0.2%, still more preferably0.025-0.175% still more preferably 0.05-0.165%, still more preferably0.075-0.15%, still more preferably 0.08-0.125%, still more preferably0.09-0.1% of the mucolytic agent (e.g., N-acetyl-L-cysteine), w/w, orany value or range of values therebetween. Most preferably, thecomposition can include (about) 0.093% N-acetyl-L-cysteine, w/w.

Visual Indicators

At least one embodiment can include a visual indicator. Preferably, thevisual indicator can be or comprise a coloring agent. More preferably,the visual indicator can be or comprise a dye or colored dye. Still morepreferably, the visual indicator can be or comprise a blue dye. Mostpreferably, the visual indicator can be or comprise FD&C Blue No. 1. Thecomposition can include a visual indicator, preferably a coloring agent,more preferably a colored dye, still more preferably a blue dye, stillmore preferably FD&C Blue No. 1.

In some embodiments, the visual indicator can be in, have, comprise, orbe provided in a dry, solid, powdered, anhydrous, and/or granular form.In some embodiments, the visual indicator can have a purity of at least,up to, and/or about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%. 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, or 99.9% (as measured by a suitable material assay, suchas CoA). In some embodiments, the visual indicator can comprise or be(provided) in the form of a stock (solution (e.g., in water)) having anysuitable concentration (e.g., about 0.01%, 0.05%, 0.075%, 0.1%, 0.125%,0.15%, 0.175%, 0.2%, 0.25%, 0.3%, or 0.5%, w/w, aqueous solution (e.g.,in water). In some embodiments, stock solution can be made using thedry, solid, powdered, anhydrous, and/or granular material. In someembodiments, the visual indicator can have a purity substantiallycorresponding to the concentration of the acid in solution (e.g., about0.2%, w/w) (as measured by a suitable material assay, such as CoA).

The visual indicator (e.g., FD&C Blue No. 1) can be included in thecomposition in any visually suitable amount, such as about 0.00037% w/w,or in a range of about 0.00005% to about 0.001%, preferably about0.0001% to about 0.00075%, more preferably about 0.0002% to about0.0005%, w/w, still more preferably about 0.0003% to about 0.0004%, w/w.In some embodiments, the composition can include a visible (or visiblysuitable) amount of a visual indicator, preferably a coloring agent,more preferably a colored dye, still more preferably a blue dye, stillmore preferably FD&C Blue No. 1. Most preferably, the composition caninclude (about) 0.00037% w/w of FD&C Blue No. 1.

In at least one embodiment, the visual indicator (e.g., FD&C Blue No. 1)can be added to the composition as a concentrate. The concentrate can bean aqueous or water-based concentrate in some embodiments. In someembodiments, the composition can include 0.01-2.5%, w/w, of a 0.01-5%,w/w (in water) visual indicator concentrate. Preferably, the compositioncan include 0.05-1%, w/w, of a 0.05-1%, w/w (in water) visual indicatorconcentrate. More preferably, the composition can include 0.075-0.5%,w/w, of a 0.075-0.5%, w/w (in water) visual indicator concentrate. Stillmore preferably, the composition can include 0.1-0.25%, w/w, of a0.1-0.25%, w/w (in water) visual indicator concentrate. Still morepreferably, the composition can include (about) 0.185% w/w of (about)0.2% w/w (in water) visual indicator concentrate. In at least oneembodiment, the visual indicator (e.g., FD&C Blue No. 1) can be includedin the composition at about 0.185%, w/w, of a ˜0.2% stock (aqueous)solution, or equivalent thereof. Most preferably, the composition caninclude (about) 0.185% w/w of (about) 0.2% w/w (in water) FD&C Blue No.1 concentrate.

Antimicrobials

In some embodiments, the composition can include an antimicrobial agent.In some embodiments, one or more of the foregoing components can exhibitantimicrobial activity. For instance, the alcohol, chaotropic agent,surfactant, and/or mucolytic agent can be antimicrobial or exhibitantimicrobial activity in some embodiments. Accordingly, certainembodiments need not include a separate antimicrobial (e.g.,bactericidal and/or bacteriostatic) agent. In one or more embodiments,the antimicrobial properties of alcohol (e.g., SDA 3C) persist even atthe lower concentrations in which the alcohol is provided in saidembodiment(s) of the present disclosure (e.g., about 17.73%, w/w, or5-25%, 10-22%, 10-20% 15-19%, 16-18.5%, 17-18.25%, or 17.5-18%, w/w, orany value or range of values therebetween).

Ribonuclease Inhibitors

Some embodiments include a ribonuclease inhibitor, or inhibitor ofribonuclease, such as heparin, heparan sulfate, oligo (vinylsulfonicacid), poly(vinylsulfonic acid), oligo(vinylphosphonic acid), andpoly(vinylsulfonic acid), or salts thereof. In certain (e.g., preferred)embodiments, the composition does not include a ribonuclease inhibitoror inhibitor of ribonuclease, or is (substantially) devoid of one ormore (e.g., any) ribonuclease inhibitor or inhibitor of ribonuclease(e.g., other than a chaotropic agent, such as guanidine thiocyanate,which may have intrinsic RNAse inhibitory activity). Accordingly, atleast one embodiment is (substantially) devoid of one or more (any)ribonuclease inhibitor, or inhibitor of ribonuclease. One or moreembodiments are (substantially) devoid of any ribonuclease inhibitor, orinhibitor of ribonuclease (e.g., other than a chaotropic agent, such asguanidine thiocyanate).

Proteases

Some embodiments include a protease. In certain (e.g., preferred)embodiments, the composition does not include a protease, or is(substantially) devoid of one or more (e.g., any) protease. Accordingly,at least one embodiment is (substantially) devoid of one or more (any)protease. Without being bound to any theory, a protease (or proteolyticenzyme, peptidase or proteinase) is a type of enzyme that breaks one ormore peptide bonds through hydrolysis, thereby converting proteins intosmaller protein fragments (or peptides) or individual protein subunits(or amino acids).

Protein Denaturants

Some embodiments include one or more protein denaturants. For instance,in at least one embodiment, the (i) chaotropic agent can be, comprise,or function as a protein denaturant (or denature proteins or have orexhibit protein denaturation activity). In at least one embodiment, the(ii) surfactant/detergent can be, comprise, or function as a proteindenaturant (or denature proteins or have or exhibit protein denaturationactivity). In at least one embodiment, the (iii) alcohol can be,comprise, or function as a protein denaturant (or denature proteins orhave or exhibit protein denaturation activity). In at least oneembodiment, the (iv) mucolytic agent can be, comprise, or function as aprotein denaturant (or denature proteins or have or exhibit proteindenaturation activity), such as when the protein(s) contain accessibledisulfide bonds or bridges. In some embodiments, two or more of the (i)chaotropic agent, (ii) surfactant/detergent, (iii) alcohol, and (iv)mucolytic agent can be, comprise, or function as a protein denaturant(or denature proteins or have or exhibit protein denaturation activity).In some embodiments, each or all of the (i) chaotropic agent, (ii)surfactant/detergent, (iii) alcohol, and (iv) mucolytic agent can be,comprise, or function as a protein denaturant (or denature proteins orhave or exhibit protein denaturation activity).

Without being bound to any theory, the protein denaturation activity ofone or more of the foregoing components or ingredients can beconcentration and/or time dependent.

Formulations

An embodiment of the present disclosure includes a nucleic acidpreservation composition (or formulation), comprising a carrier, achaotropic agent, a buffering agent, a chelating agent, a surfactant, analcohol, an acid, and a mucolytic agent. An embodiment further includesan optional visual indicator. An embodiment can include 20-50%chaotropic agent, w/w, 1-5% buffering agent, w/w, 0.05-2.5% chelatingagent, w/w, 0.05-2.5% surfactant, w/w, 5-25% alcohol, w/w, 0.005-0.25%mucolytic agent, w/w, acid qs to pH 6.5-9.5, and the carrier qs to 100%.An embodiment can further include 0.005-2.5%, w/w, visual indicator.

In at least one embodiment, the composition includes about 43.92% w/w ofthe chaotropic agent, about 2.65% w/w of the buffering agent, about0.81% w/w of the chelating agent, about 0.279% w/w of the surfactant,about 17.73% w/w of the alcohol, about 0.093% w/w of the mucolyticagent; the acid qs to a pH of about 8.0 (e.g., about 1.08% of a 37% acidsolution, or equivalent thereof), and the carrier qs to 100%. Thecomposition can include about 0.00037% w/w of the visual indicator.

In some embodiments, the carrier can be or comprise an aqueous carrier,such as water, preferably filtered, purified, distilled, and/ordeionized water. In some embodiments, the chaotropic agent can be orcomprise guanidine and/or thiocyanate, preferably guanidine thiocyanate.In some embodiments, the buffering agent can be or comprisetris(hydroxymethyl)aminomethane (Tris), preferably Tris-HCl, morepreferably Trizma® base. In some embodiments, the chelating agent can beor comprise ethylenediaminetetraacetic acid (EDTA), preferably EDTAdisodium (salt) dihydrate. In some embodiments, the surfactant can be orcomprise sodium lauroyl sarcosinate (SLS). In some embodiments, thealcohol can be or comprise a specially denatured alcohol (SDA) or amixture of ethanol and isopropanol, preferably a mixture of about 95%ethanol, v/v and about 5% isopropanol, v/v, or SDA 3C. In someembodiments, the acid can be or comprise hydrochloric acid. In someembodiments, the mucolytic agent can be or comprise N-acetyl-L-cysteine.

An embodiment of the present disclosure includes a nucleic acidstabilization and/or preservation composition, comprising about 43.92%chaotropic agent (e.g., guanidine thiocyanate), w/w, about 2.65%buffering agent (e.g., Tris), w/w, about 0.81% chelating agent (e.g.,EDTA or EDTA disodium (salt) dihydrate), w/w, about 0.279% surfactant(e.g., SLS), w/w, about 17.73% alcohol (e.g., SDA 3C), w/w, about 0.093%mucolytic agent (e.g., N-acetyl-L-cysteine), w/w, acid (e.g.,hydrochloric acid) qs to about pH 8.0; and/or a carrier (e.g., anaqueous carrier comprising filtered, purified, distilled, and/ordeionized water) qs to 100%. An embodiment can further include about0.00037%, w/w, visual indicator (e.g., FD&C Blue No. 1).

An embodiment of the present disclosure includes 43.92% chaotropic agent(e.g., guanidine thiocyanate), w/w, ±10%, 2.65% buffering agent (e.g.,Tris), w/w, ±10%, 0.81% chelating agent (e.g., EDTA or EDTA disodium(salt) dihydrate), w/w, ±10%, 0.279% surfactant (e.g., SLS), w/w, ±10%,17.73% alcohol (e.g., SDA 3C or a mixture of 95% ethanol, v/v, ±10%, and5% isopropanol, v/v, ±10%), w/w, ±10%, 0.093% mucolytic agent (e.g.,N-acetyl-L-cysteine), w/w, ±10%, and/or acid (e.g., hydrochloric acid)qs to pH 8.0, ±10%, with a carrier (e.g., an aqueous carrier, preferablyfiltered, purified, distilled, and/or deionized water) qs to 100%. Anembodiment further includes 0.00037%, w/w, ±10% visual indicator (e.g.,FD&C Blue No. 1) or equivalent thereof (e.g., 0.185%, w/w, ±10%, of a0.2%, w/w, ±10% visual indicator concentrate (e.g., in water)). In anembodiment, the amount of each component, ±10%, is further (limited tothe recited amount) ±9%, preferably ±8%, more preferably ±7%, still morepreferably ±6%, still more preferably ±5%, still more preferably ±4%,still more preferably ±3%, still more preferably ±2%, still morepreferably ±1%.

In at least one embodiment, the composition includes about 43.92%guanidine thiocyanate, w/w, about 2.65% Tris, w/w, about 0.81% EDTA orEDTA disodium (salt) dihydrate, w/w, about 0.279% SLS, w/w, about 17.73%SDA 3C, w/w, about 0.093% N-acetyl-L-cysteine, w/w, about 1.08%hydrochloric acid 37%, w/w, or equivalent thereof, or hydrochloric acidqs to a pH of about 8.0, and water qs to 100%, w/w. The composition caninclude about 0.00037% w/w of FD&C Blue No. 1 (or 0.185% w/w of a 0.2%w/w (in water) concentrate thereof), and about 32.602% water, w/w.

In some embodiments, the composition can be substantially free or devoidof microbial (e.g., bacterial, fungal, and/or viral) contamination. Insome embodiments, the composition can have less than or equal to (about)100 cfu/g bacteria or bacterial contamination. In some embodiments, thecomposition can have less than or equal to (about) 99, 98, 97, 96, 95,90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or 5cfu/g bacteria or bacterial contamination. In some embodiments, thecomposition can have less than or equal to (about) 100 cfu/g fungus (orfungi, such as yeast and/or mold) or fungal contamination. In someembodiments, the composition can have less than or equal to (about) 99,98, 97, 96, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25,20, 15, 10, or 5 cfu/g fungus (or fungi, such as yeast and/or mold) orfungal contamination. As used herein, “cfu/g” refers to colony formingunits (of the one or more microbes) per gram (of the (final and/orliquid) composition).

An illustrative embodiment of the present disclosure is presented inTable 1, below. Table 1 describes ingredients of the illustrativecomposition, as well as the use, function, and/or activity of saidingredients.

TABLE 1 Ingredients % w/w Purified water—Carrier, base solvent for anaqueous solution 32.602 Guanidine thiocyanate—Chaotropic agent; solidform. Both 43.92 guanidinium and thiocyanate ions can be chaotropic;this makes this agent superior to guanidinium chloride (chloride ion isnot chaotropic). A chaotropic agent may disrupt (denature) proteinstructure, aid in releasing protein-bound DNA, lyse cells and virusparticles, and denature nucleases, which can damage DNA (and RNA).Tris/Trizma base—Buffering agent; tris(hydroxymethyl)aminomethane; 2.65solid form, alternatively 40% (w/w) solution in water. EDTA—Chelatingagent; ethylenediaminetetraacetic acid disodium salt 0.81 dihydrate;solid form. Complexes transition metal ions that are essential forcatalyzing DNA (and RNA) degradation by nucleases. In addition, it hasantibacterial activity. SLS—Surfactant/detergent; Sodium LauroylSarcosinate; ~30% 0.93 aqueous stock solution (in water). Alternativelyin granulated form. A surfactant may lyse cells, including contaminatingmicrobes (e.g., bacteria), denature proteins, and allow release ofnucleic acids. We found that this detergent to be substantially moresoluble in our compositions than the more popular sodium dodecyl sulfate(SDS). SDA 3C—Specially Denatured Alcohol (i.e., ethanol, 95%) 3C 17.73(isopropanol, 5%). Alcohols may lyse cells, including contaminatingmicrobes (e.g., bacteria) and/or denature proteins. FDC Blue No.1—Visual indicator/coloring agent/dye; ~0.2%, w/w, 0.185 concentrate (inwater). Adds light blue color. It is not essential for DNAstabilization. It aids customer visualization of saliva mixing withstabilizing solution. Predominantly cosmetic. HCl—Hydrochloric acid;~37% w/w, stock solution (in water); ~12M. 1.08 Acids may be used toadjust pH of DNA stabilizing solution (e.g., to about 8.0 and/or wherethe nucleic acid (DNA) is most stable. N-Acetyl-L-cysteine—Mucolyticagent; solid form. Mucolytic agents 0.093 may aid in denaturing proteins(e.g., by reducing or cleaving disulfide bridges). In addition,ingredients or components (e.g., chemicals or agents) containing freesulfhydryl groups may act as antioxidants and/or may help controldissolved oxygen in the DNA Stabilizing Solution. Batch Total 100%

Table 1.1 presents another illustrative formulation for a composition ofthe present disclosure.

TABLE 1.1 Ingredients % w/w Purified water 34.12 Guanidine thiocyanate43.92 Tris/Trizma base 2.65 EDTA (disodium salt dihydrate) 0.81 SLS0.279 SDA 3C 17.73 FDC Blue No. 1 0.00037 HCl 0.4 N-Acetyl-L-cysteine0.093 Batch Total 100%

Additional features of the present disclosure can be learned from U.S.Pat. No. 7,482,116, the entirety of which is incorporate by referenceherein.

Kits

Some embodiments include a kit, such as a biological sample preservationkit. In particular, in one or more embodiments, the inventivecomposition can be incorporated into a kit. Kits can include, forexample, a composition, as disclosed and/or described herein, and asample collection apparatus. In at least one embodiment, the compositioncan be disposed in a portion of a sample collection apparatus.Illustrative sample collection apparatus can include a container or vial(e.g., a tube) having a sample collection portion. For instance, thecontainer can comprise an outer wall at least partially bounding aninternal compartment. The internal compartment can contain thecomposition, to which a biological sample can be added. Alternatively,the sample can be added to the compartment and the composition added tothe sample post-collection. For instance, the apparatus can include acomposition dispenser for adding the composition to the compartment,pre- or post-sample collection. In at least one embodiment, thedispenser can comprise a cap for closing or sealing an opening of theapparatus. The opening can lead into or be in fluidic communication withthe compartment. The cap can have a compartment for retaining thecomposition until it is to be added to the compartment of the container.

Some embodiments can include a kit comprising a biological samplecollection device (or container) and a composition of the presentdisclosure. In at least one embodiment, the composition can be disposedin a portion of the device. For instance, in some embodiments, thecomposition can be disposed in a portion of a cap or lid of the device.The collection device (or container) can be configured to receive thebiological sample (e.g., in an inner compartment thereof) and have thecomposition added thereto.

In some embodiments, the composition in the kit can be substantiallyfree or devoid of microbial contamination (as described above).

Various sample collection apparatus are described in the followingapplications, the entirety of each of which is incorporated herein byspecific reference: U.S. application Ser. No. 14/952,712, filed Nov. 25,2015; U.S. Provisional Application Ser. No. 62/370,630, filed Aug. 3,2016; U.S. Provisional Application Ser. No. 62/453,459, filed Feb. 1,2017; U.S. Provisional Application Ser. No. 62/510,174, filed May 23,2017; U.S. Provisional Application Ser. No. 62/512,594, filed May 30,2017; U.S. Provisional Application Ser. No. 62/513,235, filed May 31,2017; U.S. Provisional Application Ser. No. 62/529,355, filed Jul. 6,2017; U.S. application Ser. No. 15/667,228, filed Aug. 2, 2017;International Application Serial No. PCT/US2017/045352, filed Aug. 3,2017; U.S. application Ser. No. 15/692,259, filed Aug. 31, 2017; andU.S. Provisional Application Ser. No. 62/590,165, filed Nov. 22, 2017.

Compositions of the present disclosure can be incorporated intoapparatus described in any of the foregoing applications. Embodiments ofthe present disclosure can include a kit comprising a composition, asdisclosed and/or described herein, and a sample collection apparatusdescribed in any of the foregoing applications.

Methods of Manufacture

Some embodiments include a method of manufacturing a composition of thepresent disclosure. Embodiments can include providing or obtaining acarrier, as described herein. Embodiments can include adding to thecarrier a suitable amount of one or more components or ingredientsdescribed herein (e.g., to a final concentration described herein).Embodiments can include adding to the carrier a described amount ofstock solution of one or more components or ingredients describedherein.

At least one embodiment includes adding to the carrier a chaotropicagent, buffering agent, chelating agent, surfactant, alcohol, acid,and/or mucolytic agent. One or more embodiments can include adding tothe carrier a visual indicator. At least one embodiment includes addingto a (liquid) carrier, chaotropic agent to a final concentration of20-50%, w/w, buffering agent to a final concentration of 0.1-5%, w/w,chelating agent to a final concentration of 0.01-5%, w/w, surfactant toa final concentration of 0.01-5%, w/w, alcohol to a final concentrationof 5-25%, w/w, acid to pH 6.5-9.5 or equivalent amount, and/or mucolyticagent to a final concentration of 0.005-0.25%, w/w. At least oneembodiment includes adding to a (liquid) carrier visual indicator to afinal concentration of 0.00005-0.5%, w/w. The carrier can be included atqs to 100%

At least one embodiment includes adding to a (liquid) carrier,chaotropic agent to a final concentration of (about) 43.92%, w/w,buffering agent to a final concentration of (about) 2.65%, w/w,chelating agent to a final concentration of (about) 0.81%, w/w,surfactant to a final concentration of (about) 0.279%, w/w, alcohol to afinal concentration of (about) 17.73%, w/w, acid to pH (about) 6.5-9.5or to a final concentration of (about) 0.4%, w/w, and/or mucolytic agentto a final concentration of (about) 0.093%, w/w. At least one embodimentincludes adding to a (liquid) carrier visual indicator to a finalconcentration of (about) 0.00037%, w/w. The carrier can be included at(about) 34.12% or qs to 100%.

In some embodiments, the chaotropic agent can be or comprise guanidineand/or thiocyanate, the buffering agent can be or comprise Tris orTrizma base, the chelating agent can be or comprise EDTA or EDTAdisodium (salt) dihydrate, the surfactant can be or comprise SLS, thealcohol can be or comprise ethanol and/or isopropanol (e.g., SDA 3C),the mucolytic agent can be or comprise N-acetyl-L-cysteine, the acid canbe or comprise HCl, the carrier can be or comprise water, and/or theoptional visual indicator can be or comprise FD&C Blue No. 1.

A method of manufacturing a nucleic acid stabilization and/orpreservation composition can include adding the carrier to a vessel(e.g., charging a mixing tank with (filtered, deionized, etc.) water. Insome embodiments, the carrier can be included at a final concentrationof about 34.12%, w/w, of the composition or to qs 100%.

In some embodiments, a mixer can be activated before one or moreadditional components or ingredients are added to the carrier. In someembodiments, a mixer can be activated after one or more additionalcomponents or ingredients are added to the carrier. In some embodiments,a mixer can be set to a speed setting of 2-8, preferably 3-7, morepreferably 4-6, still more preferably 5 and/or sweep setting of 2-8,preferably 3-7, more preferably 4-6, still more preferably 5. In someembodiments, the carrier can be heated to a suitable mixing temperaturebefore one or more additional components or ingredients are added to thecarrier. In some embodiments, the carrier can be heated to a suitablemixing temperature after one or more additional components oringredients are added to the carrier. In some embodiments, the suitablemixing temperature can be (about) 55-95±5° F., preferably 60-90±5° F.,more preferably 65-85±5° F., still more preferably 70-80±5° F., mostpreferably 75±5° F.

In some embodiments, a suitable amount of chaotropic agent (e.g.,guanidine thiocyanate) can be added to the carrier (e.g., to a finalconcentration of about 43.92%, w/w of the composition). In someembodiments, the chaotropic agent can be mixed for a period of time(e.g., between 30-300 minutes, preferably 60-240 minutes, morepreferably 120-180, still more preferably 140-160 minute, mostpreferably 150 minutes, or until the chaotropic agent is dissolved (insolution) in the carrier.

In some embodiments, a suitable amount of buffering agent (e.g., Tris orTrizma Base) can be added to the carrier (e.g., to a final concentrationof about 2.65%, w/w of the composition). In some embodiments, thebuffering agent can be mixed in for a period of time (e.g., between 1-90minutes, preferably 5-60 minutes, more preferably 10-45, still morepreferably 12-30 minute, still more preferably 15-25 minute, mostpreferably (about) 20 minutes, or until the buffering agent is dissolved(in solution) in the carrier.

In some embodiments, a suitable amount of chelating agent (e.g., EDTA,EDTA disodium salt, EDTA disodium (salt) dihydrate) can be added to thecarrier (e.g., to a final concentration of about 0.81%, w/w of thecomposition). In some embodiments, the chelating agent can be mixed infor a period of time (e.g., between 1-90 minutes, preferably 5-60minutes, more preferably 10-45, still more preferably 12-30 minute,still more preferably 15-25 minute, most preferably (about) 20 minutes,or until the chelating agent is dissolved (in solution) in the carrier.In at least one embodiment, the buffering agent and the chelating agentcan be added to the carrier together, at (approximately) the same time,contemporarily, concomitantly, and/or (substantially) concurrently (orsimultaneously), with or without being pre-mixed together. In someembodiments, the buffering agent and the chelating agent can be added tothe carrier separately.

In some embodiments, a suitable amount of surfactant (e.g., SLS) can beadded to the carrier (e.g., to a final concentration of about 0.279%,w/w of the composition). In some embodiments, the surfactant can bemixed in for a period of time (e.g., between 1-90 minutes, preferably5-60 minutes, more preferably 10-45, still more preferably 15-35 minute,still more preferably 20-30 minute, most preferably (about) 25 minutes,or until the surfactant is dissolved (in solution) in the carrier.

In some embodiments, a suitable amount of alcohol (e.g., ethanol, amixture of ethanol and another chemical, such as isopropanol, or a SDA,preferably SDA 3C) can be added to the carrier (e.g., to a finalconcentration of about 17.73%, w/w of the composition). In someembodiments, the alcohol can be mixed in for a period of time (e.g.,between 5-90 minutes, preferably 10-75 minutes, more preferably 15-60,still more preferably 25-45 minute, still more preferably 30-40 minute,most preferably (about) 35 minutes, or until the alcohol is dissolved(in solution) in the carrier.

In some embodiments, a suitable amount of an optional visual indicator(e.g., a coloring agent, a dye, preferably a blue dye, such as FD&C BlueNo. 1) can be added to the carrier (e.g., to a final concentration ofabout 0.00037%, w/w of the composition). In some embodiments, the visualindicator can be mixed in for a period of time (e.g., between 5-90minutes, preferably 10-60 minutes, more preferably 15-45, still morepreferably 10-30 minute, still more preferably 15-25 minute, mostpreferably (about) 20 minutes, or until the alcohol is dissolved (insolution) in the carrier.

In some embodiments, a suitable amount of an acid (e.g., hydrochloricacid) can be added to the carrier (e.g., to a final concentration ofabout 0.4%, w/w of the composition or to a pH 8.0 of the composition).In some embodiments, the acid can be mixed in for a period of time(e.g., between 5-90 minutes, preferably 10-60 minutes, more preferably15-45, still more preferably 10-30 minute, still more preferably 15-25minute, most preferably (about) 20 minutes, or until the acid isdissolved (in solution) in the carrier and/or the mixture equilibratesat the desired pH.

In some embodiments, a suitable amount of a mucolytic agent (or reducingagent) (e.g., N-Acetylcysteine, N-acetyl-L-cysteine) can be added to thecarrier (e.g., to a final concentration of about 0.093%, w/w of thecomposition). In some embodiments, the acid can be mixed in for a periodof time (e.g., between 5-90 minutes, preferably 10-60 minutes, morepreferably 15-45, still more preferably 10-30 minute, still morepreferably 15-25 minute, most preferably (about) 20 minutes, or untilthe acid is dissolved (in solution) in the carrier and/or the mixtureequilibrates at the desired pH.

A series of illustrative manufacturing batch procedures are present inTable 3.

TABLE 3 Process Parameter Batch #1 Batch #2 Batch #3 Batch #4 3.1Addition of Water Mixing Speed (mixer/sweep) 5/5 4/4 6/6 5/5 3.2Addition of Guanidine Thiocyanate Mixing Speed (mixer/sweep) 5/5 4/4 6/65/5 Mixing Time 150 min 120 min 180 min 150 min 3.3 Addition of TrizmaBase and Disodium EDTA Addition Temperature 70° F. 65° F. 75° F. 70° F.Mixing Speed (mixer/sweep) 5/5 4/4 6/6 5/5 Mixing Time ≥75 min ≥60 min≥90 min ≥75 min 3.4 Addition of Sodium Lauroyl Sarcosinate and SDA 3CMixing Speed (mixer/sweep) 5/5 4/4 6/6 5/5 Mixing Temperature 75 ± 5° F.70° F. 80° F. 75 ± 5° F. Mixing Time 25 min 20 min 30 min 25 min 3.5Addition of Hydrochloric Acid Mixing Speed (mixer/sweep) 5/5 4/4 6/6 5/5Mixing Temperature 75 ± 5° F. 70° F. 80° F. 75 ± 5° F. Mixing Time 20min 15 min 25 min 20 min 3.6 Addition of Color Concentrate Mixing Speed(mixer/sweep) 5/5 4/4 6/6 5/5 Mixing Temperature 75 ± 5° F. 70° F. 80°F. 75 ± 5° F. Mixing Time 20 min 15 min 25 min 20 min 3.7 Addition ofN-Acetylcysteine Mixing Speed (mixer/sweep) 5/5 4/4 6/6 5/5 MixingTemperature 75 ± 5° F. 70° F. 80° F. 75 ± 5° F. Mixing Time 45 min 30min 60 min 45 min

Quality control testing can be performed at any suitable point duringmanufacture. For example, upon completion of the bulk manufacturingprocess for each batch, two (2) samples (approximately 4 ounces each)were aseptically obtained from the bulk blend tank using clean andsanitized, approved and appropriate tools for obtaining samples fromeach of the following locations: top surface of batch near center oftank, top surface of batch near side wall of tank, middle of batch nearcenter of tank, middle of batch near side wall of tank, bottom of batchnear center of tank, and bottom of batch near side wall of tank. Eachsample was placed in a sterile cup and labeled.

Each sample was tested for proper appearance, specific gravity, and pH.In addition, assays were performed to test concentration and/oreffectiveness of the chelating agent, alcohol, and mucolytic agent. Inaddition, contamination (microbial limits) were tested by measuringtotal aerobic plate count, yeast and mold, Staphylococcus aureus, andPseudomonas aeruginosa. Table 4 presents testing specifications forvarious quality control measures.

TABLE 4 TEST METHOD SPECIFICATION Appearance SOP 403 Comparable toStandard Specific gravity @ 25° C. SOP 405 Report only pH STM M4037.9-8.3  Assay - Disodium EDTA Cornerstone 0.73-0.89% Assay - SDAAlcohol 3C Cornerstone 15.96-19.50% Assay - N-Acetylcysteine Cornerstone0.084-0.102% Microbial limits STM M429 Less than 100 cfu/g Yeast andmold STM M429 Less than 100 cfu/g Staphylococcus aureus STM M429 AbsencePseudomonas aeruginosa STM M429 Absence

In some embodiments, the method can include sealing the composition in asuitable storage vessel or a portion of a sample collection apparatus(e.g., a composition storage portion of a container or vial (e.g., atube). Samples were also subjected to controlled room temperature (CRT)and accelerated (ACC) stability testing in storage vessels and samplecollection apparatus.

In some embodiments, the method can produce or result in a compositionthat can be substantially free or devoid of microbial contamination (asdescribed above).

Methods of Use

Some embodiments include a method of preserving and/or stabilizingnucleic acid. The method can comprise providing a biological samplecontaining the nucleic acid and combining a composition of the presentdisclosure with the biological sample. In at least one embodiment, thebiological sample can be a mucin-containing bodily fluid or tissue, suchas sputum or saliva. The method can include reducing the viscosity of amucin-containing bodily fluid or tissue (e.g., by reducing disulfidebonds inherent to mucin with a mucolytic agent or reducing agent).

In at least one embodiment, the nucleic acid is DNA. In someembodiments, the composition can stabilize the nucleic acid or DNA(e.g., against degradation). In some embodiments, the composition canstabilize the nucleic acid or DNA for a first period of time. In someembodiments, the first period of time can be greater than or equal toabout 14 days, 30 days, 60 days, 90 days, 120 days, 240 days, 300 days,or 365 days. In some embodiments, the composition can stabilize thenucleic acid or DNA for the first period of time at room temperature,between −20° C. to 50° C., or other suitable temperature or temperaturerange. In some embodiments, the composition can be stable for a secondperiod of time. In some embodiments, the second period of time can begreater than or equal to about 12 months, 18 months, 24 months, 30months, or 36 months. In some embodiments, the composition can bestabile for the second period of time at room temperature, between −20°C. to 50° C., or other suitable temperature or temperature range.

At least one embodiment includes a method of recovering a nucleic acidfrom sputum, comprising: i) obtaining sputum from a subject, ii)contacting the sputum with a composition of the present disclosure toform a sample mixture, iii) optionally contacting the mixture with aprotease, and iv) recovering the nucleic acid from the mixture.

In some embodiments, the composition does not significantly inhibit orinterfere with subsequent nucleic acid analysis, such as DNAamplification (via PCR), (next generation) sequencing, and so forth,when added in a suitable amount to the biological sample.

Sample Collection

Some embodiments of the present disclosure include obtaining, providing,and/or collecting a biological sample (e.g., from a subject, such as ahuman subject). In some embodiments, the biological sample can be orcomprise (human) saliva. The (human) sample can be collected aseptically(to avoid (microbial) contamination). In one or more embodiments, thesample can be collected into a sample collection apparatus or samplecontainer thereof. In some embodiments, the sample collection apparatusor container can be part of a kit and/or can include a composition ofthe present disclosure. Embodiments can include contacting the samplewith a composition of the present disclosure.

DNA Extraction and Analysis:

Some embodiments of the present disclosure include extracting nucleicacid from the biological sample. The following is a non-exhaustivelisting or description of various modes of extraction or extractionprocedures that may be suitable for use with compositions of the presentdisclosure.

Extraction Chemistry

Organic—Phenol chloroform extraction is still a mechanism employed inboth research and clinical labs and is sample type dependent when itcomes to tissue source. A manual phenol/chloroform extraction followedby a chloroform back extraction to help remove any organic solventcontamination will be performed to extract high molecular weight genomicDNA.

Salting out—Both home brew and commercial salting out chemistries arewidely used for high molecular weight nucleic acid extraction. Theapproach requires a high concentration of salt be added to the salivasample in order to crash out nucleic acid under the addition of ethanol.A series of washes are performed to remove excess salt from the sampleprior to analysis.

Solid phase—A variety of technology providers offer both spin column andvacuum manifold solutions for binding DNA to a solid support for nucleicacid purification. Once the DNA is attached to the support a series ofwashes are performed. Ultimately DNA is eluted off of the solid supportin a small volume for analysis. Spin column chemistry is frequently usedin both the research and clinical lab.

Bead-based—Beads or (para)magnetic beads are prepared with variousbinding moieties or by charge in order to bind high molecular weightDNA. The beads are captured by a magnetic field so anything unbound tothe beads can be washed away as part of the purification process. Oncewashing is complete the nucleic acid is eluted off of the beads with asolution that solubilizes the DNA leaving the beads behind which aresubsequently removed by reapplying a magnetic field. There are bothsmall and large volume automated solutions for this approach in theresearch and clinical environment.

Illustrative Extractions

Ten DNA samples previously extracted from the saliva collection kitscontaining compositions of the present disclosure and up to six samplesfrom an existing saliva collection kit were used for testing. Anadditional 23 samples were newly collected using the inventive salivacollection kit. Each of the 23 samples were extracted in duplicate 700ul aliquots. Standard QC was performed to assess the quality of the DNA.

23/23 samples were extracted with two replicates per sample. Averagecombined yield by UV spectrophotometry (Nanodrop) of all samples was20.4 ug (2.8-111.4). 20/23 extractions had 260/280 ratios above thedesired value of 1.7, although the three samples lower than 1.7 arelikely to perform well in downstream analysis. All samples had highmolecular weight DNA, as shown in FIG. 1A.

700 ul of saliva sample solution was extracted using Perkin Elmerreagents for the MSM1 (Chemagen) extraction system. Concentrations forall of the samples were determined by UV spectrophotometry (Nanodrop).An estimate of purity was determined with UV spectrophotometry bymeasuring the A260/A280 absorbance ratio. Additionally, samples wereanalyzed on an agarose gel to visualize sample integrity. A molecularweight sizing ladder (L) and a control sample of greater than 50 kb (C)are included on each gel. Bionexus All Purpose HI-LO DNA Marker Used onQualitative Gels (see FIG. 3B).

Analytical Approaches

Some embodiments include analyzing the extracted nucleic acids. Severalmethods are available for analyzing the extracted nucleic acids. Thefollowing is a non-exhaustive listing or description of various methodsfor analyzing the extracted nucleic acids that may be suitable for usewith compositions of the present disclosure.

PCR

Polymerase Chain Reaction (PCR) analysis is a rapid and cost effectivemeans for assessing the fidelity and cleanliness of DNA templates. Aseries of PCR reactions (of varying size amplicons) will be generatedfrom all DNA templates and resolved via electrophoresis for the correctsize amplification product. The range of PCR amplicon sizes will provideinformation on the fidelity of all DNA extraction products.

qPCR

Quantitative PCR (qPCR) uses dual labeled fluorogenic probes for thequantitation of PCR amplicons. Allelic discrimination utilizing Taqmanchemistry will be used to determine the specific genotype for all DNAscollected and extracted across all extraction approaches. Genotypes foreach of the subjects will be measured for concordance across allvariables being analyzed. All quantitative measurements will be made intriplicate.

dPCR

Digital PCR (dPCR) is an emerging technology being employed forsensitive detection of genotypes in samples with limiting amounts and/orlimiting quality. The same Taqman assays will be used to determine theabsolute sensitivity of every DNA sample extracted. Given thesensitivity of dPCR we will be able to determine the ultimatesensitivity of each variant being analyzed.

Microarray

The measurement of hundreds of thousands or millions of SNPssimultaneously has tremendous implications when it comes to bothdiscovery and clinical classification of a single DNA sample. Thesensitivity and specificity requirements are quite different than QPCRbased analysis and the approach for SNP detection is also different asthis analytical approach uses a hybridization based mechanism foridentifying DNA variants. Call rates and SNP concordance across donorsprocessed with different DNA extraction chemistries will be a criticalanalytical endpoint.

Sanger Sequencing

The gold standard for variant analysis will be employed across allsamples in this study. The target regions for analysis will cover thesame amplicons of QPCR, dPCR and Microarray to cross validate thegenotypes across all other analytical methods. The ability to make highquality sanger base calls (and hence variants) is highly dependent onthe quality of nucleic acid. This approach is used regularly forclinical analysis.

NextGen Sequencing

As used herein, “next generation sequencing” (NGS), also known ashigh-throughput sequencing, refers to non-Sanger-based, high-throughputDNA sequencing technologies. Through NGS, millions or even billions ofDNA strands can be sequenced in parallel, yielding substantially morethroughput and minimizing the need for the fragment-cloning methods thatare often used in Sanger sequencing of genomes. NGS is the catch-allterm used to describe a number of different modern sequencingtechnologies or platforms including, for example, pyrosequencing,sequencing by synthesis, sequencing by ligation, ion semiconductorsequencing, and others as known in the art.

As understood by those skilled in the art, NGS generally allowsequencing of large amounts of DNA and RNA much more quickly andaffordably than Sanger sequencing. In NGS, vast numbers of short readsare sequenced in a single stroke. To do this, firstly the input samplecan be cleaved into short sections. The length of these sections dependson the particular sequencing machinery used. Illustrative examples ofspecific NGS technologies include, for example, Illumina® (Solexa)sequencing, Roche 454™ sequencing, Ion Torrent™: Proton/PGM sequencing,SOLiD sequencing, and so forth.

In Illumina sequencing, 100-150 bp reads are used. Somewhat longerfragments are ligated to generic adaptors and annealed to a slide usingthe adaptors. PCR is carried out to amplify each read, creating a spotwith many copies of the same read. They are then separated into singlestrands to be sequenced. The slide is flooded with nucleotides and DNApolymerase. These nucleotides are fluorescently labelled, with the colorcorresponding to the base. They also have a terminator, so that only onebase is added at a time. An image is taken of the slide. In each readlocation, there will be a fluorescent signal indicating the base thathas been added. The slide is then prepared for the next cycle. Theterminators are removed, allowing the next base to be added, and thefluorescent signal is removed, preventing the signal from contaminatingthe next image. The process is repeated, adding one nucleotide at a timeand imaging in between. Computers are then used to detect the base ateach site in each image and these are used to construct a sequence. Allof the sequence reads will be the same length, as the read lengthdepends on the number of cycles carried out.

Roche 454™ sequencing can generally sequence much longer reads thanIllumina®. Like Illumina®, it does this by sequencing multiple reads atonce by reading optical signals as bases are added. As in Illumina®, theDNA or RNA is fragmented into shorter reads, in this case up to 1 kb.Generic adaptors are added to the ends and these are annealed to beads,one DNA fragment per bead. The fragments are then amplified by PCR usingadaptor-specific primers. Each bead is then placed in a single well of aslide. So each well will contain a single bead, covered in many PCRcopies of a single sequence. The wells also contain DNA polymerase andsequencing buffers. The slide is flooded with one of the four NTPspecies. Where this nucleotide is next in the sequence, it is added tothe sequence read. If that single base repeats, then more will be added.So if we flood with Guanine bases, and the next in a sequence is G, oneG will be added, however if the next part of the sequence is GGGG, thenfour Gs will be added. The addition of each nucleotide releases a lightsignal. These locations of signals are detected and used to determinewhich beads the nucleotides are added to. This NTP mix is washed away.The next NTP mix is now added and the process repeated, cycling throughthe four NTPs. This kind of sequencing generates graphs for eachsequence read, showing the signal density for each nucleotide wash. Thesequence can then be determined computationally from the signal densityin each wash. All of the sequence reads we get from 454 will bedifferent lengths, because different numbers of bases will be added witheach cycle.

Unlike Illumina® and Roche 454™, Ion Torrent™ and Ion proton sequencingdo not make use of optical signals. Instead, they exploit the fact thataddition of a dNTP to a DNA polymer releases an H+ ion. As in otherkinds of NGS, the input DNA or RNA is fragmented, this time ˜200 bp.Adaptors are added and one molecule is placed onto a bead. The moleculesare amplified on the bead by emulsion PCR. Each bead is placed into asingle well of a slide. Like Roche 454™, the slide is flooded with asingle species of dNTP, along with buffers and polymerase, one NTP at atime. The pH is detected is each of the wells, as each H+ ion releasedwill decrease the pH. The changes in pH allow us to determine if thatbase, and how many thereof, was added to the sequence read. The dNTPsare washed away, and the process is repeated cycling through thedifferent dNTP species. The pH change, if any, is used to determine howmany bases (if any) were added with each cycle.

Additionally, or alternatively, the sequencing may be more generallyperformed by a fluorescent-based sequencing technique and/or anyelectrical-current-based sequencing technique. Illustrative examples offluorescent-based sequencing techniques include any technique thatincorporates nucleotides conjugated to a fluorophore, such as, forexample sequencing using Illumina® based sequencing methods and systems.Illustrative examples of electrical-current-based sequencing techniquesinclude any sequencing technique (including strand sequencing methods)that measures the electrical current of a polynucleotide as it passesthrough a pore inserted into a charged membrane or otherwisespecifically disrupts the electrical current of a sensor and/or chargedmembrane. A non-limiting example of electrical-current-based sequencingtechniques include the Nanopore DNA sequencing systems and methods ofOxford NanoPore Technologies®.

Strand sequencing systems, such as those provided by Oxford NanoPoreTechnologies®, provide some advantages when determining copy numbervariation of a nucleic acid, particularly the copy number variation of asample that potentially contains DNA (or other nucleic acid) fromneoplastic and/or cancerous cells. For example, in strand sequencingtechniques, a single portion of the genome is continuously sequenced,which allows a direct analysis of copy number variation instead of animplicit analysis of copy number variation that may occur when analyzingsequencing data provided by other sequencing methods where the samplenucleic acid is cut into small fragments for sequencing. This may beparticularly advantageous for embodiments when sequence coverage is low.That is, in some embodiments, a low sequence coverage run may return anincomplete set of genomic data. It may be possible to infer from thesequence data the presence and/or absence of genomic regions in additionto an implicit copy number for each sequenced region. However, in astrand sequencing method, the long sequence reads produced may allow fora more definitive assessment of copy number variation, particularly forregions that are duplicated or deleted. If a full sequence is notavailable due to the low coverage of the sequencing run, it may bedifficult to determine what portions of the genome are deleted (a formof copy number variation) versus what portions of the genome were notrepresented based on statistical probability (i.e., random sampling).

As an illustrative example, a sequencing run that generates data having0.5× coverage will theoretically leave half of the sample unrepresented.Using sequencing methods that “chop up” the nucleic acid into smallfragments for sequencing, the final product may be a sequence libraryrepresenting about half of the total reference genome, where an alignedreference genome is littered with a smattering of smaller nucleic acidmatches. On the other hand, using a strand sequencing method, again atlow coverage (e.g., 0.5×), the result may be a sequence libraryrepresenting, again, about half of the total reference genome. However,when aligned with a reference genome, the matching portions are muchlonger and may provide more definitive information, such as whatsequences have been deleted, duplicated, inserted, etc. This may alsoprove problematic. While a longer contiguous portion of the genome maybe represented by a strand sequencing approach, long contiguous portionsof the genome are also left unknown. So, although strand sequencingmethods may allow for a higher definition view of portions of thegenome, smaller sequencing reads have the potential to provide a moreglobal picture of the entire genome. In this and other ways, strandsequencing may provide a robust model for analyzing copy numbervariation.

Though the foregoing is illustrative of known sequencing techniques andtheir applications to the inventive methods and systems disclosedherein, it should be understood that this does not preclude as yetundiscovered or otherwise undisclosed sequencing methods from beingapplied within the scope of the present invention. That is, thesequencing method, itself, is not, in many embodiments, a requisiteinventive step (unless, for example, an improvement is provided to themethod and/or system through use of a particular sequencing technique);rather, what is done with the sequencing data provided by the sequencingmethod and/or how those data are applied generally comprises aninventive step. Accordingly, it should be appreciated that futuresequencing technologies (and those sequencing technologies that have notbeen explicitly listed herein), if used as a tool in the disclosedmethod or systems, are included within the scope of this application.

Additionally, any of the foregoing sequencing techniques may be used inany number or capacity and with any number of flow cells or othersimilar inputs that affect the total number of sequencing reads providedfor each sequencing reaction/run.

Next Generation sequencing may ultimately become the standard foranalysis of both DNA and RNA targets. A targeted panel including thegenomic regions covered by qPCR, dPCR and array based targets is createdfor all DNA samples through a standard library preparation process.Samples are barcoded and multiplexed on a NextGen platform for variantanalysis. Data is de-multiplexed and analyzed for direct comparison ofgenotype call across all other platforms.

Several of the above and other DNA-based downstream methods were testedto assess the quality and usability of DNA extracted from samplescollected using 2 mL saliva collection kits containing a nucleic acidpreservative composition of the present disclosure. Additionally, asmall number of DNA samples, extracted from two existing products wereused for comparison for some downstream methods. Below is anon-exhaustive listing or description of several downstream methodstested, including TaqMan® chemistry for detection of single nucleotidepolymorphisms (SNPs) using an OpenArray® format (n=120 SNPs/sample), acopy-number variant (CNV) using TaqMan® chemistry (CYP2D6 gene), wholeexome next-generation sequencing (WES) (Thermo Fisher) and chromosomalmicroarray analysis (CMA) (Affymetrix CytoScan HD). These methods werechosen to include a wide variety of common methods used in moleculargenetics laboratories. In addition to the downstream analysis, thebacterial DNA content as a percentage of total DNA was measured using aquantitative PCR (qPCR) assay. Without being bound to any theory, salivasamples are known have high concentration of bacterial DNA that could bean interfering substance for some methods.

TaqMan® Open Array® SNP Genotyping

Genotyping for the single nucleotide polymorphism was accomplished usinga TaqMan® OpenArray® genotyping assay. The TaqMan® assay is an allelediscrimination assay using PCR amplification and a pair of fluorescentdye detectors that target the SNP. One fluorescent dye is attached tothe detector that is a perfect match to the first allele (e.g. an “A”nucleotide) and a different fluorescent dye is attached to the detectorthat is a perfect match to the second allele (e.g. a “C” nucleotide).During PCR, the polymerase will release the fluorescent probe intosolution where it is detected using endpoint analysis in a LifeTechnologies, Inc. Specifically OpenArray® technology is a nanoliterfluidics platform for low-volume solution-phase reactions. TheOpenArray® technology uses a microscope slide-sized plate with 3,072through holes. Each through-hole is 300 μm in diameter and 300 μm deepand is treated with hydrophilic and hydrophobic coating. TaqMan®chemistry for a single assay is preloaded and dried down in each throughhole. OpenArrays® were obtained through Life Technologies design andmanufacturing. Genotypes were determined using Life Technologies' TaqmanGenotyper v1.0.1 software.

A total of 5234 genotypes were determined on 44 samples on a 118-120SNPs/sample. The 44 samples included repeats of 3 samples each fromextractions from both the inventive and existing kits. Genotyping ofsamples from the inventive kits was highly successful and exceeded knowperformance expectations for this type of assay. Without being bound toany theory, Taqman genotyping is expected to successfully yieldgenotyping on greater than 99% of samples. In this experiment, 99.75% ofsamples produced a genotype (5221/5234). There were no significantdifferences in genotyping rate between the inventive solution DNAextracts and the existing extracts, 99.74% and 99.87%, respectively. Inthe 6 samples duplicated in both the inventive solution DNA extracts andthe existing extracts, all genotypes were concordant.

Taqman® Copy-Number Variant Detection

A TaqMan® Copy Number Assay (CYP2D6-Hs00010001_cn) was used to detectthe copy number of the CYP2D6 gene, a well characterized CNV evaluatedin pharmacogenetics. TaqMan® Copy Number Assays employ TaqMan® MGB probechemistry to evaluate the copy number of genomic DNA targets. This assayused an Applied Biosystems® 7900 HT real-time PCR instruments and copycaller software to determine the copy number. Each sample was amplifiedthree times and plotted against a standard curve to determine copynumber.

33 inventive extracted DNA samples and 5 existing extracted samples wereanalyzed for a well-characterized copy number variant in the CYP2D6gene. 30/33 inventive solution extracted DNA samples produced CNVresults. 5/5 competitor extracted samples produced CNV results. The 3samples that did not produce a CNV result were all from the same person(“B”) from 3 independent samples collected on the same day. A samplefrom this same individual that was collected on a different day andextracted from the existing saliva kit did produce a normal CNV resultruling out a potential interfering mutation.

Whole Exome Sequencing (WES)

An exome library was prepared using Ion AmpliSeg™ Exome Kit. The librarykit is combined with Ampliseq Exome Panel Primer pools, which containsapproximately 294,000 primers pairs across 12 primer pools. The targetedresulting amplicons are then treated with a reagent to partially digestthe primers and phosphorylate the amplicons. The amplicons are thenligated to Ion Adapters with barcodes and purified. Upon the completionof the exome library preparation, the purified, exome-enriched libraryis quantified by real-time PCR. The quantified library is then dilutedto 100 pM and used to prepare templated Ion PI™ Ion Sphere™ Particles(ISPs) for sequencing. The sample was then sequenced on the Ion ProtonSystem using an Ion PI™ Chip v3. Ion Hi-Q Sequencing 200 V2 chemistrywas used to sequence up to 200-base pair average insert libraries.

Four samples, 3 extracted from the inventive saliva kit and oneextracted from the existing saliva kit were evaluated with a whole exomelibrary prep (AmpliSeq Exome, Thermo Fisher) followed by next generationsequencing on the Ion Proton instrument (Thermo Fisher). Typicallyexpected results are >30 million reads, mean depth of coverage ofgreater than 80× and >80% of bases covered at a depth of >20×. Three offour samples met these criteria. There was one inventive saliva kitextracted sample that did not meet two of the three QC metrics having<30 million reads and less that 80× mean depth coverage. It is notedthat the underperforming sample was one of the same samples that alsodid not have a successful CNV analysis. Examination of the DNA QCprofile did point to anything unusual about this sample. All QC metricswere met. Although underperforming this sample yielded adequate exomesequencing results for evaluation.

Chromosomal Microarray (CMA)

The CMA analysis was conducted using the Affymetrix CytoScan HD assayfollowing the manufacturer's protocol. The samples were scanned on aGenome Analyzer 3000. Chromosomal microarrays were used to detectchromosomal aberrations at a higher resolution than karyotyping. Theassay consisted of DNA preparation followed by hybridization to theCytoScan HD chip that contains approximately 2.7 million CNVs across thegenome. The samples were evaluated using the Affymetrix ChAS software.

One sample was selected from the Spectrum saliva kit extracted DNAs. Itwas successfully evaluated on a chromosomal microarray (Affymetrix,CytoScan HD). The sample had a MAPD value of ≤0.25 (0.18), SNPQC valueof ≥15 (16.47), a waviness value of ≤0.12 (0.09) and a QC call rate of≥95% (96.8%).

Bacterial DNA Content Using a qPCR Assay

Bacterial DNA content with in the sample was determined using a modifiedprotocol described in the literature. Briefly, a standard curve wascreated using a serial dilution of E. coli to compare to real time PCRdata generated. PCR primers were chosen from a region of the 16S rRNAgene that is known to be conserved across a wide variety ofmicroorganisms and is not found in eukaryote DNA. The DNA was tested forthe presence of the 16S rRNA gene using real-time qPCR on a ThermoFisher7900HT instrument using copy caller software.

Bacterial DNA content, as a percentage of the total amount of DNA fromthe saliva collected sample, has been thought to possibly inhibit orreduce the success rate of the downstream analysis. 33 DNA samplesextracted from the inventive saliva kit and 5 DNA samples extracted fromthe existing saliva kit were tested for the percentage of bacterial DNApresent. Previous data from the competitor estimated the percentage ofbacterial DNA to be approximately 13%. The average bacterial content ofthe inventive saliva kit extractions was 5.5% (1.1-14.3%). The averagebacterial content of the competitor saliva kit extractions was 26%(2.1-96.2%)-14.31%).

A series of the above and/or other experimental tests were performed tosupport an FDA submission for 510K consideration in order to obtainapproval for use of a formulation of the present disclosure in acollection device for nucleic acid extraction using any one of a varietyof available chemistry approaches, including organic, solid-phase,bead-based, and salting out extraction, as well as any one of a varietyof currently used molecular analysis, including PCR, qPCR, dPCR,microarray, Sanger sequencing, and so-called next generation (orNextGen) sequencing (NGS), as outlined in Table 5, below.

TABLE 5 Extraction Chemistry/ Analytical Micro- Sanger NextGen PlatformPCR QPCR dPCR array Sequencing Sequencing Organic X X X X X XSolid-phase X X X X X X Bead-based X X X X X X Salting out X X X X X XSummary of Results

DNA was successfully extracted from all samples in all replicates. Ingeneral, the size (and yield) of the extracted DNA was high. There wasminimal evidence of degradation. The replicates from a sample were verycomparable in terms of yield. Additionally, the yields across what areassumed to be the same individual behaved similarly. Genotyping for SNPsproduced a high quality result and met expected yield. Samples from oneindividual, 4 separately collected samples, did not meet QC metrics forthe CNV (n=3) or NGS (n=1). Bacterial DNA content as a percentage of thetotal DNA was relatively low in the inventive saliva kit DNAextractions.

In a further example, presented in Tables 6 and 7, 100 samples were usedto test the performance of a nucleic acid preservative composition ofthe present disclosure (“Inventive”) and two existing products(“Existing 1” and “Existing 2”). As presented in Table 6, the“Inventive” nucleic acid preservative composition of the presentdisclosure yielded a higher average concentration of DNA and a higheramount (yield) of total DNA than either of the “Existing” products.

TABLE 6 Average Average Average Kit Conc. (ng/ul) Volume (ul) Yield (ug)Existing 1 124.50 460.44 47.32 Inventive 152.26 436.23 62.05 Existing 2145.42 438.59 58.16

As further presented in Table 7, samples processed with the “Inventive”nucleic acid preservative composition of the present disclosure had asignificantly lower average amount of non-human DNA than either of the“Existing” products.

TABLE 7 Average Overall Average Average Average Non-Human Viscosity Kit260/230 260/280 FQC % DNA % Score Existing 1 1.30 1.72 96.7 9.3 5Inventive 0.83 1.79 98.9 4.5 3 Existing 2 1.08 1.71 95.6 11.2 6

Accordingly, compositions of the present disclosure are surprisingly,significantly superior to existing DNA preservation products. Inparticular, it was surprising and unexpected that the compositions ofthe present disclosure work so well (e.g., yield high amounts of (human)nucleic acid (DNA) and/or have or exhibit low levels of microbialcontamination). It was further surprising and unexpected that thecompositions of the present disclosure work so well with the low amountof alcohol provided in some embodiments. For instance, in someembodiments, the amount of alcohol included in the composition can beless (e.g., about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% less)than typical, traditional, or existing nucleic acid preservationsolutions. In addition, the lower amount of alcohol of more economicaland/or makes the composition more amendable to shipping or transport(e.g., by more easily complying with shipping requirements andregulations, reducing volatility, etc.).

CONCLUSION

It will be appreciated that certain embodiments (e.g., compositions,kits, method, etc.) may include, incorporate, or otherwise comprisefeatures (e.g., properties, components, ingredients, elements, parts,portions, steps, etc.) described in other embodiments disclosed and/ordescribed herein. Accordingly, the various features of one embodimentcan be compatible with, combined with, included in, and/or incorporatedinto other embodiments of the present disclosure. Disclosure of certainfeatures relative to one embodiment of the present disclosure should notbe construed as limiting application or inclusion of said features tothe specific embodiment. Rather, it will be appreciated that otherembodiments can also include said features without necessarily departingfrom the scope of the present disclosure. Moreover, unless a feature isdescribed as requiring another features in combination therewith, anyfeature described herein may be combined with any other feature of asame or different embodiment disclosed herein.

The described embodiments are to be considered in all respects only asillustrative and not restrictive. The scope of the invention is,therefore, indicated by the appended claims rather than by the foregoingdescription. All changes which come within the meaning and range ofequivalency of the claims are to be embraced within their scope. Variousalterations and/or modifications and additional applications of thefeatures illustrated herein which would occur to one skilled in therelevant art and having possession of this disclosure, can be made tothe illustrated embodiments without departing from the spirit and scopeof the invention as defined by the claims, and are to be consideredwithin the scope of this disclosure. While various features andembodiments have been disclosed herein, other features and embodimentsare contemplated. For instance, well-known features and embodiments arenot described herein in particular detail in order to avoid obscuringaspects of the described embodiments. Such features and embodiments are,however, also contemplated herein.

The entirety of International PCT Application Serial No.PCT/US2018/013862, filed Jan. 16, 2018, entitled “Nucleic AcidPreservation Solution and Methods of Manufacture and Use,” isincorporated herein by reference.

I claim:
 1. A human nucleic acid preservation composition, comprising:39.53%-48.31% guanidine thiocyanate, w/w; 2.39%-2.92%tris(hydroxymethyl)aminomethane (Tris), w/w; 0.73%-0.89%ethylenediaminetetraacetic acid (EDTA) or EDTA disodium (salt)dihydrate, w/w; 0.251%-0.307% sodium lauroyl sarcosinate (SLS), w/w;15.96%-19.50% alcohol, w/w, the alcohol comprising ethanol;0.084%-0.102% N-acetyl-L-cysteine (NAC), w/w; hydrochloric acid qs to pH7.5-8.5; and an aqueous carrier comprising filtered, purified,distilled, and/or deionized water.
 2. The composition of claim 1 furthercomprising a dye or coloring agent.
 3. The composition of claim 1,wherein the composition comprises: 43.48%-44.36%, w/w, of the guanidinethiocyanate; 2.62%-2.68%, w/w, of the Tris; 0.80%-0.82%, w/w, of theEDTA or EDTA disodium (salt) dihydrate; 0.276%-0.282%, w/w, of the SLS;17.55%-17.91%, w/w, of the alcohol; 0.092%-0.094%, w/w, of the NAC; andthe hydrochloric acid qs to pH 7.8-8.2.
 4. The composition of claim 1,wherein the composition is substantially free or devoid of mucolyticagent(s) and/or reducing agent(s) other than N-acetyl-L-cysteine.
 5. Thecomposition of claim 1, wherein the composition comprises:41.72%-46.12%, w/w, of the guanidine thiocyanate; 2.52%-2.78%, w/w, ofthe Tris; 0.77%-0.85%, w/w, of the EDTA or EDTA disodium (salt)dihydrate; 0.265%-0.293%, w/w, of the SLS; 16.84%-18.62%, w/w, of thealcohol; 0.088%-0.098%, w/w, of the NAC; and The acid qs to pH 7.6-8.4.6. The composition of claim 1, wherein the composition is substantiallyfree or devoid of antimicrobial agent(s), bactericidal agent(s), and/orbacteriostatic agent(s) other than the guanidine thiocyanate, SLS,alcohol, and NAC.
 7. The composition of claim 1, wherein the compositionis substantially free or devoid of inhibitor(s) of ribonuclease otherthan the guanidine thiocyanate, SLS, alcohol, and NAC.
 8. Thecomposition of claim 1, wherein the composition is substantially free ordevoid of protease(s).
 9. The composition of claim 1, wherein thealcohol comprises a mixture of about 95% ethanol, v/v, and about 5%isopropanol, v/v.
 10. A nucleic acid preservation composition,comprising: a carrier; 20-50%, w/w, of a chaotropic agent; 1-5%, w/w, ofa buffering agent; 0.05-2.5%, w/w, of a chelating agent; 0.05-2.5%, w/w,of a surfactant; 5-25%, w/w, of alcohol; an acid qs to pH 7.2-8.8; and0.005-0.25%, w/w, of a mucolytic agent.
 11. The composition of claim 10,wherein the composition comprises: 39.53%-48.31%, w/w, of the chaotropicagent; 2.39%-2.92%, w/w, of the buffering agent; 0.73%-0.89%, w/w, ofthe chelating agent; 0.251%-0.307%, w/w, of the surfactant;15.96%-19.50%, w/w, of the alcohol; 0.084%-0.102%, w/w, of the mucolyticagent; and the acid qs to pH 7.5-8.5%.
 12. The composition of claim 11,wherein the composition is substantially free or devoid of antimicrobialagent(s), bactericidal agent(s), and/or bacteriostatic agent(s) otherthan the chaotropic agent, the surfactant, the alcohol, and themucolytic agent.
 13. The composition of claim 11, wherein thecomposition is substantially free or devoid of inhibitor(s) ofribonuclease other than the chaotropic agent, the surfactant, thealcohol, and the mucolytic agent.
 14. The composition of claim 11,wherein the composition is substantially free or devoid of protease(s).15. The composition of claim 10, wherein the composition comprises:43.48%-44.36%, w/w, of the chaotropic agent; 2.62%-2.68%, w/w, of thebuffering agent; 0.80%-0.82%, w/w, of the chelating agent;0.276%-0.282%, w/w, of the surfactant; 17.55%-17.91%, w/w, of thealcohol; 0.092%-0.094%, w/w, of the mucolytic agent; and the acid qs topH 7.8-8.2.
 16. The composition of claim 15, wherein the composition issubstantially free or devoid of antimicrobial agent(s), bactericidalagent(s), and/or bacteriostatic agent(s) other than the chaotropicagent, the surfactant, the alcohol, and the mucolytic agent.
 17. Thecomposition of claim 15, wherein the composition is substantially freeor devoid of inhibitor(s) of ribonuclease other than the chaotropicagent, the surfactant, the alcohol, and the mucolytic agent.
 18. Thecomposition of claim 15, wherein the composition is substantially freeor devoid of protease(s).
 19. The composition of claim 10, wherein: Thecarrier comprises filtered, purified, distilled, and/or deionized water;the chaotropic agent comprises guanidine thiocyanate; the bufferingagent comprises tris(hydroxymethyl)aminomethane (Tris); the chelatingagent comprises ethylenediaminetetraacetic acid (EDTA) the surfactantcomprises sodium lauroyl sarcosinate (SLS); the alcohol comprises theacid comprises hydrochloric acid (HCl); and the mucolytic agentcomprises N-Acetyl-L-cysteine (NAC).
 20. The composition of claim 10,furthering comprising a dye or coloring agent.
 21. The composition ofclaim 10, wherein the composition is substantially free or devoid ofmucolytic agent(s) and/or reducing agent(s) other thanN-acetyl-L-cysteine.
 22. The composition of claim 10, wherein thecomposition comprises: 41.72%-46.12%, w/w, of the chaotropic agent;2.52%-2.78%, w/w, of the buffering agent; 0.77%-0.85%, w/w, of thechelating agent; 0.265%4293%, w/w, of the surfactant; 16.84%-18.62%,w/w, of the alcohol; 0.088%-0.098%, w/w, of the mucolytic agent; and Theacid qs to pH 7.6-8.4.
 23. The composition of claim 10, wherein thecomposition is substantially free or devoid of antimicrobial agent(s),bactericidal agent(s), and/or bacteriostatic agent(s) other than thechaotropic agent, the surfactant, the alcohol, and the mucolytic agent.24. The composition of claim 10, wherein the composition issubstantially free or devoid of inhibitor(s) of ribonuclease other thanthe chaotropic agent, the surfactant, the alcohol, and the mucolyticagent.
 25. The composition of claim 10, wherein the composition issubstantially free or devoid of protease(s).